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Am J Physiol Lung Cell Mol Physiol 291: L837-L846, 2006. First published June 2, 2006; doi:10.1152/ajplung.00134.2006
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IL-12 overexpression in mice as a model for Sjögren lung disease

Sharon McGrath-Morrow,1,* Beth Laube,1,* Shey-Cherng Tzou,2 Cecilia Cho,1 Jeffrey Cleary,1 Hiroaki Kimura,2 Noel R. Rose,2,3 and Patrizio Caturegli2,3

1Department of Pediatrics and 2Department of Pathology, The Johns Hopkins School of Medicine; and 3Department of Molecular Microbiology and Immunology, The Johns Hopkins School of Public Health, Baltimore, Maryland

Submitted 11 April 2006 ; accepted in final form 19 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Interleukin-12 (IL-12), a Th1 proinflammatory cytokine, is reported to be increased in Sjögren syndrome. To evaluate the effects of local Th1/Th2 deregulation, we generated a transgenic mouse model that overexpresses IL-12 in the lungs. IL-12 transgenic mice developed bronchial and alveolar abnormalities strikingly similar to those found in the lungs of Sjögren patients. Pathologically, lung abnormalities began at ~4 mo of age and were characterized by lymphocytic infiltrates around the bronchi, intraluminal periodic acid Schiff-positive debris, increased cell proliferation in the alveolar region, and increased interstitial and alveolar macrophages. Functionally, these abnormalities translated into decreased mucociliary clearance (P < 0.05 vs. wild-type littermates) and increased oxidative stress (P < 0.01). The pathological and functional abnormalities were accompanied by significant changes in lung natural killer (NK) cells. The number of NK cells was fourfold higher in IL-12 transgenic than wild-type lungs (20% of all lymphoid cells vs. 5%) during the first month of life. NK cells then decreased within a narrow window of time (from 30 to 50 days of age), reaching a nadir of ~2% on day 50, and remained at these low levels thereafter. This new mouse model highlights the role of IL-12 in the initiation of Sjögren syndrome.

autoimmunity; transgenic mice; inflammation; natural killer cells

SJÖGREN SYNDROME is a systemic autoimmune disease that involves lacrimal glands (dry eyes), salivary glands (dry mouth), and numerous extraglandular sites such as lungs, kidneys, skin, and thyroid (18). In its primary form, Sjögren syndrome is defined as the presence of the above manifestations without additional connective tissue disease. In its secondary form, Sjögren syndrome associates with other rheumatic autoimmune diseases, mainly rheumatoid arthritis, systemic lupus erythematosus, or systemic sclerosis. Like most rheumatic diseases, Sjögren syndrome lacks a single distinguishing feature and therefore its diagnosis relies on a combination of clinical and laboratory findings. In particular, the revised American-European classification identifies the following six criteria: ocular symptoms, oral symptoms, ocular signs, lymphocytic infiltration of salivary glands on lower lip biopsy, objective evidence of salivary gland involvement, and antibodies to SSA and/or SSB ribonucleoprotein antigens. To establish the diagnosis of Sjögren syndrome, the patient must have either lymphocytic infiltration of salivary glands or SSA/SSB antibodies, plus any other three of the remaining criteria (54). Sjögren syndrome is more common in women (female-to-male ratio is 9:1), often occurring in the fifth or sixth decade of life, and has an estimated population prevalence of 0.5% (53). Therefore, on the basis of the Census 2000 estimate of the total US population (281,421,906), Sjögren syndrome affects ~1.4 million Americans.

Lung abnormalities are frequent in patients with Sjögren syndrome (56). Davidson (13) found them in ~25% of patients, but the frequency increases to 50% when thin-section chest computed tomography is used for diagnosis (51). Patients present with irritating dry cough (secondary to dryness of the tracheobronchial mucosa, tracheobronchial sicca) (11), dyspnea from small airway obstruction (39, 42), and increased bronchial responsiveness (20, 45). The clinical course is insidious and invariably requires the use of glucocorticoids. Histological changes in the lungs are numerous and are still awaiting a better classification (4), although all have lymphocytic infiltration in common. When the infiltrate is diffuse, interstitial lesions are described as lymphocytic interstitial pneumonia, usual interstitial pneumonia, and nonspecific interstitial pneumonia, all considered distinct pathological subsets. When the infiltrate becomes confluent, often with a lobar distribution, the terms "bronchiolitis obliterans organizing pneumonia" (BOOP), and more recently "cryptogenic organizing pneumonia" have been used (53). In addition to the above-described interstitial disease, other studies have also emphasized the involvement of the tracheobronchial airways (30) and an impaired mucociliary clearance (35). Patients with Sjögren syndrome can also develop tumors of the mucosa-associated lymphoid tissue present in the lungs (12). Finally, it is not unusual for patients with Sjögren syndrome to develop both autoimmune hypothyroidism and lung dysfunctions (18). Regarding this association, it is important to clarify that although nonautoimmune hypothyroidism can also be associated with lung abnormalities (55), these abnormalities are different from the inflammatory lung response seen in the autoimmune hypothyroidism associated with Sjögren syndrome (5).

Interleukin-12 (IL-12) is a proinflammatory cytokine that has been found to be increased in Sjögren syndrome, in both the serum and the organs targeted by the autoimmune attack (27, 29, 38, 41). IL-12 is a heterodimer (p70) composed of a p40 subunit (expressed predominantly by macrophages, dendritic cells, and granulocytes) and a p35 subunit (expressed constitutively in numerous cell types); both subunits must be expressed by the same cell to produce a bioactive molecule. IL-12 binds to a specific plasma membrane receptor, mainly expressed on activated T, B, and natural killer (NK) lymphocytes. The receptor is composed of a beta1-subunit, which binds p40, and a beta2-subunit, which binds p35. Ligation of the IL-12 receptor activates Tyk2 and Jak2 kinases, which, in turn, induce phosphorylation, dimerization, and nuclear translocation of signal transducer and activator of transcription-4 (3). The outcome is the transcriptional activation of numerous genes involved in prototypic Th1 responses, such as interferon-{gamma}. The classic functions of IL-12 are to control parasitic infections and cancer cells by inducing an inflammatory response. However, uncontrolled IL-12 levels can induce a damaging lymphocyte and macrophage infiltration in many organs such as the lungs (10, 15). For this reason, we hypothesized that prolonged elevated levels of IL-12 could lead to many of the pathological changes observed in patients with Sjögren syndrome. We tested this hypothesis with a IL-12 transgenic mouse model previously developed in our laboratories (26).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mouse population. The study used IL-12 transgenic mice that express the full-length IL-12 heterodimer in the thyroid gland (26). These mice develop local features of thyroid disease (primary hypothyroidism and lymphocytic infiltration of the thyroid) but also produce enough IL-12 that IL-12 is increased in the serum (26) and can presumably exert effects at sites distant from the thyroid. IL-12 transgenic mice were bred to wild-type CBA mice (purchased from Jackson Laboratory, Bar Harbor, ME) for at least eight generations and maintained as heterozygous. A total of 187 mice, 123 transgenic mice between 41 and 689 days old and 64 age-matched controls, were analyzed in this study. In particular, 138 mice (98 transgenic and 40 control) were assessed for salivary, lacrimal, and lung histopathology; 13 mice (6 transgenic and 7 control) for lung IL-12 content, immunohistochemistry, immunofluorescence, and mucociliary clearance; and 36 mice (19 transgenic and 17 control) for lung NK cells. All experiments were conducted in accordance with the standards established by the United States Animal Welfare Acts, set forth in National Institutes of Health guidelines, and approved by the Johns Hopkins University Animal Care and Use Committee.

Lung content of IL-12 and other cytokines. Lungs filter the majority of the cardiac output and contain abundant heparan sulfate proteoglycans, which are known to bind IL-12 (22). Lungs should therefore demonstrate increased IL-12 concentrations in response to increased serum IL-12 levels. To prove this prediction, proteins were extracted from the lungs of IL-12 transgenic and control mice as follows. The left lung lobes were removed and minced into small pieces (~5 mm in diameter) in 3 ml of phosphate-buffered saline (PBS) containing a cocktail of protease inhibitors (Sigma, St Louis, MO). Lung pieces were homogenized by ice-cold mechanical disruption with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) and centrifuged at 1000 g for 10 min and 4°C to remove undisrupted debris and nuclei. The supernatant was then ultracentrifuged (100,000 g for 1 h at 4°C) to separate the soluble cytosolic fraction from the pellet (formed by intracellular organelles). After the protein concentration was measured by bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL), the cytosolic fraction was used immediately for cytokine array experiments (see below) or stored at –80°C until use. Lung cytosolic proteins were diluted 1:10 and incubated overnight onto a mouse cytokine array (RayBiotech, Norcross, GA), which allows the simultaneous detection of 31 molecules distributed as follows: 11 cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-17, IFN-{gamma}, and TNF-{alpha}), 11 chemokines {CCL2 [monocyte chemoattractant protein (MCP)-1/JE], CCL3 [macrophage inflammatory protein (MIP)-1], CCL5 (RANTES), CCL11 (Eotaxin), CCL12 (MCP-5), CCL17 [thymus- and activation-regulated chemokine (TARC)], CCL19 (MIP-3), CCL21 (6Ckine), CCL27 (CTAK), CXCL1 (KC), and CXCL2 (MIP-2)}; 5 hematopoietic promoting molecules (IL-3, granulocyte colony-stimulating factor, granulocyte colony-stimulating factor, stem cell factor, and thrombopoietin); and 4 other molecules (leptin, tissue inhibitor of metalloproteinases 1, soluble TNF receptor 1, and vascular endothelial growth factor). After addition of biotinylated anti-cytokine antibody cocktail and horseradish peroxidase-conjugated streptavidin, a colorimetric signal was induced by addition of the detection buffer. The membrane was finally exposed to radiographic film (X-Omat AR; Kodak, Rochester, NY), and the signals were analyzed for gray scale intensity with the free software Image J (http://rsb.info.nih.gov/ij).

TGF-beta1, not present on the above-described cytokine array, was measured in isolation with a commercially available sandwich enzyme immunoassay (Quantikine; R&D, Minneapolis, MN).

Salivary, lacrimal, and lung histopathology. Lacrimal glands, salivary glands, and lungs were analyzed for the systemic effects of increased serum IL-12 concentrations.

Lacrimal and salivary glands were fixed overnight in Beckstead fixative, processed, and embedded in paraffin. Five-micrometer sections underwent hematoxylin and eosin staining. The extent of mononuclear cell infiltration was then graded with a five-category system, according to the system reported by Groom et al. (19). Briefly, a score of 0 represented a normal gland with no infiltration; 1 indicated the presence of 1–5 foci of mononuclear cells (>20 cells per focus); 2 indicated >5 foci but no parenchymal destruction; 3 indicated multiple confluent foci with moderate degeneration of parenchymal tissue; and 4 indicated extensive infiltration of the gland with mononuclear cells and severe parenchymal destruction.

Lungs were infused through the trachea with a hot (60°C) 0.5% agarose solution, under 25 cmH2O pressure, and then cooled on ice. Lungs were then fixed overnight in 10% buffered formalin, processed, embedded in paraffin, cut into 5-µm sections, and then stained with hematoxylin and eosin for further analysis. The extent of mononuclear cell infiltration was graded with a four-category system that took into account the number and the size of the bronchus-associated lymphoid tissue (BALT), as well as the interstitial infiltration: 0 represented normal lungs with no infiltration; 1, 2, and 3 indicated mild, moderate, and severe infiltration, respectively.

For periodic acid Schiff (PAS) staining, hydrated sections were oxidized in 0.5% periodic acid solution for 5 min, rinsed in water, and incubated with Schiff reagent (Sigma) for 15 min. After a final 5-min wash, slides were counterstained with Mayer's hematoxylin solution for 1 min, dehydrated, and mounted with Permount.

Lung immunohistochemistry. Immunohistochemistry was used to detect CD4+ or CD8+ T cells, B220+ B cells, MAC3+ macrophages, nitrotyrosine, and phospho-Smad2 (pSmad2).

For the hematopoietic markers (CD4, B220, and MAC3), 5-µm frozen sections were cut, mounted onto Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA), fixed in acetone, air-dried, and rehydrated in PBS. After block of endogenous peroxidase activity (5 min in 0.3% hydrogen peroxide) and antibody aspecific binding (1 h in 2% normal goat serum), sections were incubated overnight at 4°C in a humid chamber with a biotin-conjugated monoclonal antibody recognizing CD4 (clone H129.19; Pharmingen, San Diego, CA; 1:30 dilution), CD8 (Pharmingen 53-6.7; 1:30 dilution), B220 (Pharmingen RA3-6B2; 1:2,000 dilution), or MAC3 (Pharmingen 10-3.6; 1:2,000 dilution). Sections were washed in PBS and incubated with peroxidase-conjugated streptavidin (DakoCytomation, Carpinteria, CA) for 30 min at room temperature in the humid chamber. Staining was revealed by the addition of a diaminobenzidine substrate (Sigma). Finally, sections were rinsed in distilled water, counterstained with Mayer's hematoxylin (Polyscientific, Bay Shore, NY), washed in running tap water, dehydrated through increasing concentrations of ethanol, and mounted with Cytoseal (Richard-Allan Scientific, Kalamazoo, MI).

For nitrotyrosine detection, sections were first incubated in citrate buffer for 20 min (antigen retrieval), washed, and blocked in 3% H2O2 for 15 min. The specific nitrotyrosine antibody (Ab7048–50; Abcam, Cambridge, MA; 1:1,000 dilution) was applied for 30 min and then washed with PBS. The secondary antibody, InnoGenex mouse-on-mouse Iso-IHC kit (HC-3119-06; InnoGenex, San Ramon, CA), was applied for 10 min at room temperature, washed, processed as per the Vectastain Universal Quick Kit (PK-8800, Burlingame, CA), developed with diaminobenzidine (no. K3468; DakoCytomation), counterstained, dehydrated, and mounted with Permount. The brown staining was quantified with Image Pro Plus software (Media Cybernetics, Silver Spring, MD) and normalized to the lung perimeter.

For pSmad2 antibody detection, the primary antibody (no. 3101; Cell Signaling, Danvers, MA; 1:1,000 dilution) was applied for 1 h at room temperature. Secondary antibody, using Envision+ System HRP (no. K4010, DakoCytomation), was applied for 1 h at room temperature and then processed as above.

Lung immunofluorescence. Immunofluorescence was used to detect proliferating cell nuclear antigen (PCNA) as follows. Five-micrometer deparaffinized, hydrated sections were rinsed and blocked with avidin/biotin for 10 min and washed with PBS. PCNA antibody (catalog no. SC-7907; Santa Cruz, Santa Cruz, CA; 1:50 dilution) was applied for 1 h at room temperature. Sections were washed, and a secondary antibody (goat anti-rabbit Alexa Fluor 488, no. A11001; Molecular Probes, Eugene, OR; 1:200 dilution) was applied for 30 min at room temperature and washed. Sections were then incubated for 5 min with DAPI (no. D1306; Molecular Probes; 1:1,000 dilution), washed, and mounted with Vector Hard-set mount.

Mucociliary clearance. Mucociliary clearance was measured in the lungs of transgenic mice with a noninvasive procedure that involved the aspiration of a gamma-emitting radioisotope that deposited in the airway mucus and gamma camera imaging of the lungs over time. A similar approach was used previously (29, 32, 33) to quantify mucociliary clearance in humans. Briefly, mice were anesthetized for restraint purposes via an intraperitoneal injection of a ketamine-xylazine mixture (45 and 8 mg/kg, respectively). They were then suspended by their upper incisors on an inclined surface with suture material, and their tongue was gently extended with a pair of lined forceps. After the nares were occluded with tweezers, 50 µl of saline containing the gamma-emitting isotope 99mtechnetium (99mTc)-sulfur-colloid were pipetted into the distal part of the oropharynx. With the tongue extended and the nares closed, the mice were unable to swallow and aspirated the liquid containing the radioisotope into the lower respiratory tract. This aspiration procedure, which is similar to that described by Foster and colleagues (17), was repeated once to ensure adequate dosing for imaging purposes. The total amount of 99mTc-sulfur-colloid that was aspirated by the mice was ~80 µCi. 99mTc-sulfur-colloid was chosen for these experiments because it has a half-life of ~6 h and is insoluble. Therefore, it either remained in the mucus of the lungs or was removed from the lungs over time via the mucociliary escalator.

Images of the site where the radioisotope deposited in the lungs were acquired for 4 min immediately after aspiration (time 0) and at 3 and 6 h thereafter, with an X-Spect gamma camera (Gamma Medica, Northridge, CA). Mice were allowed to awaken between imaging acquisitions and were reanesthetized with half-doses (22.5 mg/kg ketamine, 4 mg/kg xylazine) before the next acquisition. The images were later processed by computer in terms of total counts detected within the two lungs combined. Counts in both lungs combined were then background corrected. Counts detected in both lungs at 3 and 6 h after aspiration were also decay corrected to time 0.

Counts detected at time 0 within the two lungs combined were considered to reflect 100% retention of the radioactivity initially deposited. Decay-corrected counts at 3 and 6 h were expressed as a percentage of the time 0 activity. All three percent retention values were plotted versus time, and a best-fit line, determined through linear regression analysis, was calculated. Percent retention at 6 h (Y) was then recalculated, based on the liner regression equation for the best-fit line (Y = b0 + b1 * 6), where b0 is the intercept and b1 is the slope. Mucociliary clearance at 6 h was expressed on a continuous scale from 0 to 100 as the complement of percent retention and calculated from the equation: % mucociliary clearance at 6 h = 100% – Y.

Lung NK cell population. To analyze the NK cell population, lungs were flushed via intracardial injection of PBS, minced into small pieces (~1 mm3), and digested by collagenase (0.1% wt/vol in Dulbecco's modified Eagle's medium) for 1 h at 37°C. The digested samples were then passed through a sterile 70-µm nylon mesh (Sefar America, Depew, NY) to remove tissue remnants and aggregating cells. Lung cells were incubated for 5 min on ice with ACK lysis buffer (to lyse erythrocytes), washed, counted, and resuspended in flow buffer (1% bovine serum albumin and 0.05% sodium azide in PBS) at a concentration of ~10 x 106 cells/ml. Next, to reduce the nonspecific (background) staining, lung cells were incubated for 10 min on ice with an unlabeled monoclonal antibody that recognizes and blocks the extracellular domain of the murine Fc{gamma} receptor (clone 2.4G2 from Pharmingen; 0.5 µg per million cells). Lung cells were finally stained with a panel of fluorochrome-conjugated antibodies (all from Pharmingen, except where indicated), including cychrome conjugated anti-CD45 (a pan-hematopoietic marker) and fluorescein isothiocyanate-conjugated anti-CD49b (clone DX5, a marker expressed on the majority of NK cells; eBioscience, San Diego, CA), phycoerythrin-conjugated anti-CD94 (another NK cell marker), and phycoerythrin-conjugated anti-CD3 (a pan T cell marker). All staining combinations contained CD45, so that lung cells could be gated with a side scatter vs. CD45 display to distinguish CD45-negative epithelial cells from CD45-positive hematopoietic cells. Cells were counted on a FACSCalibur flow cytometer, acquiring 25,000–30,000 events per sample. Data were analyzed with CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Statistical analysis. Five outcome measures were compared between IL-12 transgenic and wild-type mice: 1) salivary, lacrimal, and lung histopathology scores; 2) lung IL-12 and TGF-beta1 contents; 3) PCNA, nitrotyrosine, pSmad2 immunohistochemical stainings; 4) percent mucociliary clearance; and 5) percentage of lung NK cells. Histopathology scores were measured on an ordinal scale and compared with an ordered logistic regression model that included genotype, age, and sex as covariates. Lung IL-12 and TGF-beta1 levels, PCNA, nitrotyrosine and pSmad2 immunohistochemical stainings, and percent mucociliary clearance were compared with the Wilcoxon rank sum test. Percentages of lung NK cells at different ages were compared with a multiple linear regression model that included genotype and age as covariates. Considering that the NK cell slopes of the two genotypes changed direction (nonlinearity) in the interval between day 30 and day 60, the covariate age was transformed into two segments with a cutpoint (knot) at day 45. All analyses were performed with Stata statistical software (release 8; Stata, College Station, TX), except for immunohistochemical stainings that were assessed with SPSS 14 statistical software (Chicago, IL). A P value ≤0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
IL-12 content is increased in lungs of transgenic mice. We extracted proteins from the lungs of six IL-12 transgenic and seven control mice and tested them on a cytokine array to measure the levels of IL-12 p70 and other inflammatory cytokines. Compared with wild-type lungs (Fig. 1A), IL-12 transgenic lungs (Fig. 1B) contained significantly greater levels of IL-12 (P < 0.0001 by Wilcoxon rank sum test; Fig. 1C). MIP-2, another inflammatory marker, was also detected in IL-12 transgenic lungs (Fig. 1B), but not in wild-type lungs. The other cytokines present on the array were not detected in either wild-type or transgenic lungs. These findings indicate that transgenic IL-12 produced in the thyroid reaches the systemic circulation and accumulates in the lungs.


Figure 1
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  		Fig. 1. Lung content of interleukin-12 (IL-12). Lung proteins were incubated on a cytokine array membrane to detect the expression of several cytokines. No clear signals were detected in wild-type lungs (A), whereas IL-12 transgenic (TG) lungs (B) showed increased levels of IL-12 (arrow) and also macrophage inflammatory protein-2 (MIP-2; arrowhead). The control signals (4 dots in bottom left corner and 2 dots in top right corner) were similar in the 2 membranes (C, open bars), whereas the IL-12 signal was significantly higher in IL-12 transgenic lungs (C, filled bars). Representative images of 3 independent experiments are shown.

 
Chronically elevated levels of IL-12 increase lymphocytic infiltration in salivary glands, lacrimal glands, and lungs. In salivary and lacrimal glands, the infiltration appeared as foci of mononuclear cells around ducts and vessels, sometimes surrounding the acinar cells (Fig. 2A), a picture reminiscent of that obtained from Sjögren patients on lip biopsy. B220-positive B lymphocytes were the most abundant infiltrating cells, followed by CD4-positive T lymphocytes (data not shown). We compared the salivary and lacrimal pathology in 98 transgenic mice, aged 41–689 days, and in 40 age-matched controls, using an ordered logistic regression model. The model showed that, with age and sex held constant, IL-12 transgenic mice had a significantly more prevalent and more severe lymphocytic infiltration than controls in both salivary (Fig. 2B) and lacrimal (Fig. 2C) glands. In particular, going from the wild-type to the IL-12 transgenic genotype, the salivary score increased 0.92 [95% confidence interval (CI): 0.12–1.72; P = 0.024] and the lacrimal score increased 1.64 (95% CI: 0.84–2.43; P < 0.0001). Lacrimal and salivary pathology was more severe in older mice: for every 100-day increase in age, the scores increased 0.92 (95% CI: 0.65–1.17; P < 0.0001), with genotype and sex held constant. Approximately 80% of IL-12 transgenic mice developed moderate to severe (grade 2 or above) inflammation of the lacrimal and salivary glands when they were 6 mo of age or older. With increasing age, rare foci of infiltrating lymphocytes could be seen in the salivary and lacrimal glands of wild-type controls, but they never surpassed grade 2 (Fig. 2, B and C). Gland pathology was more severe and prevalent in female mice, reflecting the sex bias typical of Sjögren syndrome. In particular, females had scores that were on average 0.97 higher than males (95% CI: 0.26–1.67; P = 0.007).


Figure 2
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  		Fig. 2. Salivary gland histopathology and salivary, lacrimal, and lung histopathology scores. A: foci of mononuclear infiltration in salivary glands of IL-12 transgenic mice. B–D: salivary, lacrimal, and lung histopathology scores, respectively, in entire cohort of 98 IL-12 transgenic and 40 wild-type mice.

 
In lungs, lymphocytes were predominately localized around large bronchial and pulmonary vessels, yielding a striking expansion of the BALT (Fig. 3 : compare transgenic lungs in B to wild-type lungs in A). Lymphocytes populating the BALT were mainly B220-positive B lymphocytes (Fig. 3C and Table 1), followed by CD4-positive T lymphocytes (Fig. 3D and Table 1), similar to what was observed in salivary and lacrimal glands. The lung mononuclear cell infiltration was significantly more severe in IL-12 transgenic than in wild-type littermates (Fig. 2D). In particular, going from the wild-type to the IL-12 transgenic genotype, the lung score increased 3.01 (95% CI: 2.16–3.93; P < 0.0001). The lung score worsened with age: lung alterations began at ~2 mo of age, increased in prevalence and severity by 4 mo of age, and reached 100% prevalence by 6 mo of age. For every 100-day increase in age, the score increased 0.21 (95% CI: 0.02–0.42; P = 0.048), with genotype and sex held constant. Male and female transgenic mice developed similar lung lesions.


Figure 3
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  		Fig. 3. Lung histopathology. IL-12 transgenic lungs were characterized by a marked infiltration of mononuclear cells and expansion of the bronchial-associated lymphoid tissue (B). Lymphocytes were predominantly B220+ B cells (C) and CD4+ T cells (D). A wild-type lung is shown for comparison in A.

 

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  		Table 1. Distribution of main lymphoid populations present in lungs of IL-12 transgenic and wild-type littermates

 
Macrophages are increased in IL-12 transgenic lungs. IL-12 transgenic lungs also contained numerous MAC3-positive macrophages scattered throughout the interstitium and the alveolar spaces (Fig. 4, A and B). In some areas, macrophages and epithelial cells clustered, remotely resembling granulomas (Fig. 4, B and C). Macrophages were also found in close approximation to areas of BOOP (Fig. 4D). These changes were not seen in lungs from wild-type controls (Fig. 4E).


Figure 4
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  		Fig. 4. Alveolar macrophages, bronchiolitis obliterans organizing pneumonia (BOOP) and periodic acid Schiff (PAS)-positive airway material in IL-12 transgenic lungs. A: numerous alveolar macrophages (arrow) surround a consolidated area in IL-12 transgenic lungs. B: macrophages stain positive for MAC3 (brown cells). C: area of consolidation in IL-12 transgenic lungs, remotely resembling a granuloma (black arrow). An airway containing acellular, PAS-positive material is also seen at bottom left corner (blue arrow). D: area of BOOP in small bronchiole of IL-12 transgenic lungs (arrow). E: wild-type lung. F: large airway containing a large cellular debris (bordered by the 2 arrows) in IL-12 transgenic lungs.

 
Cell proliferation and PAS staining are increased in IL-12 transgenic lungs. Because IL-12 transgenic lungs had many features consistent with an inflammatory process, we evaluated cell proliferation with PCNA immunofluorescent staining. PCNA staining (not shown) demonstrated that proliferating cells were significantly more numerous in IL-12 transgenic than wild-type lungs (P < 0.02).

As mice aged, the number of inflammatory cells in the lungs increased. Several older mice showed increased PAS staining in the large and small airways (Fig. 4C). This PAS-positive debris was acellular, suggesting an increase in mucin debris from IL-12 transgenic airways. No PAS-positive material was found in the airways of any wild-type lungs examined (Fig. 4E). Some of the IL-12 transgenic lungs also showed cells that pealed off in layers into the airways and resembled bronchial epithelial cells (Fig. 4F). The findings of PAS-positive acellular debris and increased cells in the airways prompted us to study mucociliary clearance.

Mucociliary clearance is decreased in IL-12 transgenic mice. Mucociliary clearance was measured in six IL-12 transgenic females (141 ± 14 days old) and seven wild-type females (113 ± 48 days old). Representative images of a pair of wild-type (A and C) and transgenic (B and D) mouse lungs are shown in Fig. 5. Six hours after the aspiration of the radioactive tracer, wild-type lungs (Fig. 5C) retained only 82.2% of the radioactivity deposited at time 0 (Fig. 5A), yielding a robust mucociliary clearance of 17.8%. In contrast, IL-12 transgenic lungs at 6 h (Fig. 5D) retained 94.5% of the time 0 radioactivity (Fig. 5B), yielding a mucociliary clearance of only 5.5%. Analysis of the entire data set (Fig. 5E) showed that mucociliary clearance was significantly decreased in IL-12 transgenic mice (P = 0.05). One wild-type mouse (Fig. 5E) was unusual with respect to the other wild-type animals because it showed no clearance at all. When the outlier was excluded from the analysis, a P value of 0.015 was obtained. Overall, these data suggest that the presence of IL-12 in the lungs leads to a significant decrease in mucociliary clearance, a finding similar to that described in patients with Sjögren syndrome (35).


Figure 5
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  		Fig. 5. Mucociliary clearance. A–D: gamma camera images of the lungs for a wild-type (A and C) and a IL-12 transgenic (B and D) mouse. The activity inside the orange border is the lung activity; the activity outside the orange border is the gastrointestinal activity (i.e., what was cleared by mucociliary clearance and swallowed). Mice are in the supine position with heads facing the right border, so that right lungs are at top and left lungs at bottom of each image. Wild-type lungs at 6 h (C) retained 82.2% of the radioactivity that had been deposited at time 0 (A), yielding a robust mucociliary clearance of 17.8%. In contrast, transgenic lungs at 6 h (D) still retained 94.5% of the time 0 radioactivity (B), yielding a mucociliary clearance of only 5.5%. E: results for entire data set with mucociliary clearance significantly decreased in 6 IL-12 transgenic mice compared with 7 wild-type controls (P = 0.05).

 
Oxidative stress and TGF-beta signaling are increased in IL-12 transgenic lungs. Given the increase in inflammatory cells in IL-12 transgenic lungs, we quantified oxidative stress in the lungs with an antibody directed against nitrotyrosine. IL-12 transgenic lungs (Fig. 6B) showed significantly greater nitrotyrosine staining than control lungs (Fig. 6A) (P < 0.03), suggesting a link between the development of an inflammatory phenotype and the increase in oxidative stress.


Figure 6
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  		Fig. 6. Increased nitrotyrosine and phospho-Smad2 (pSmad2) staining in IL-12 transgenic lungs. A: no nitrotyrosine staining in control lungs. B: increased nitrotyrosine staining in IL-12 transgenic lungs (brown stain). C: pSmad2 staining in control lungs (black arrows). D: increased pSmad2 staining in IL-12 transgenic lungs: staining was observed in infiltrating inflammatory cells, but also in interstitial cells of alveolar septum (arrow in higher-magnification inset at bottom right corner of D). E: quantification of nitrotyrosine staining; IL-12 lungs have significantly greater staining than control (P < 0.03 by Wilcoxon rank sum test). F: quantification of pSmad2 staining; IL-12 lungs have significantly greater staining than control (P < 0.03 by Wilcoxon rank sum test).

 
Increased oxidative stress and inflammation are described in autoimmune diseases, along with a compensatory increase in TGF-beta signaling (2). We therefore evaluated the TGF-beta signaling pathway with an antibody directed against pSmad2, a key cytoplasmic protein necessary for mediating intracellular signaling of TGF-beta (34). IL-12 transgenic lungs (Fig. 6D) showed significantly greater pSmad2 staining than wild-type lungs (Fig. 6C) (P < 0.03), present both in the inflammatory cells and in the interstitial cells of the alveolar septum (inset in Fig. 6D). We also measured the overall TGF-beta1 content in whole lung homogenates from five IL-12 transgenic and five wild-type control mice. IL-12 transgenic lungs contained higher TGF-beta1 levels (5.1 ± 0.7 vs. 3.4 ± 0.4 pg/ml), although the difference did not reach statistical significance (P = 0.11 by Wilcoxon rank sum test), probably because of the focal nature of the lung infiltrate.

Lung NK cells initially increased and then decreased in IL-12 transgenic mice. Lung NK cells were studied in 19 IL-12 transgenic mice, ranging in age from 15 to 381 days, and in 17 age-matched controls. In wild-type lungs, NK cells represented ~5% of the total lymphoid population in the first month of life, increased to ~12% during the second month of life, and then remained stable at this level thereafter (Fig. 7A). These data are in keeping with the previously reported kinetics of rodent NK cells in the spleen (23).


Figure 7
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  		Fig. 7. Kinetics of lung natural killer (NK) cells. Lung NK cells were quantified by flow cytometry, monitored over a prolonged period of time, and expressed as % of the total lung lymphoid population. A shows the contrasting kinetics of wild-type ({blacksquare}) and IL-12 transgenic ({circ}) lung NK cells. Transgenic percentages were significantly different from wild-type percentages at all time points (P < 0.0001). Numbers at top indicate the number of mice used at each time point for each genotype. B and C show representative flow cytometry displays from a pair of 4-mo-old mice: lung NK cells in IL-12 transgenic lung are ~7-fold lower than in wild type (2.5% vs. 14.3%).

 
In contrast, NK cells showed a striking biphasic kinetic in IL-12 transgenic lungs (Fig. 7A). NK cells were fourfold higher than in wild-type littermates (20% vs. 5%) during the first month of life. NK cells then decreased during a narrow window of time (from 30 to 45 days of age), reaching a nadir of ~2% around day 45 and remaining at this low level throughout their life. Lung NK cell changes were also present when data were analyzed as absolute numbers rather than percentages (data not shown). Regression analysis showed that lung NK cells in IL-12 transgenic mice were significantly different from those in wild-type controls at all time points examined (P < 0.0001).

A representative flow cytometry dot plot from a pair of 4-mo-old mice is also shown in Fig. 7, B and C; the top left quadrants identify the NK cell population, which is CD3 negative and DX5 positive. At this age, NK cells in IL-12 transgenic lungs (Fig. 7B) were sevenfold lower than in wild-type lungs (Fig. 7A) (2.5% vs. 14.3%). These data suggest that the decrease in NK cell population may be an important determinant of the lung phenotype.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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This study shows that increased IL-12 content in murine lungs induces a phenotype similar to that observed in Sjögren syndrome. The lung phenotype was mainly characterized by a marked mononuclear infiltration (lymphocytes and macrophages). With age, lungs developed striking expansion of BALT, areas of BOOP in the peripheral airways, evidence of increased oxidative stress and TGF-beta signaling, decreased mucociliary clearance, and derangement of the normal lung NK cell population. The mononuclear infiltration and the BALT expansion strongly indicate an autoimmune pathogenesis and are reminiscent of the findings observed in a well-characterized mouse model of autoimmunity: the lupus-prone MRL-FASlpr mouse (48, 52). Similar to our IL-12 transgenic mice, MRL-FASlpr mice also develop mononuclear infiltrates in lungs and salivary and lacrimal glands. Interestingly, when MRL-FASlpr mice are crossed onto a IL-12 p40-knockout background, they show an improvement in lung and salivary and lacrimal gland histopathology (25), emphasizing the role of IL-12 in the disease pathogenesis. Our findings also suggest that the bronchial airways may be targeted for inflammation in Sjögren syndrome, and this notion is supported by the observation that mononuclear peribronchial infiltrates are seen on transbronchial biopsy of Sjögren patients (42).

The impaired mucociliary clearance observed in the IL-12 transgenic mice may be explained by at least three possible mechanisms. First, IL-12 transgenic lungs contained PAS-positive, acellular debris that partially occluded the airways and may have slowed mucociliary clearance by mechanical obstruction. Interestingly, glycoprotein-containing substances, including mucins, stain positive for PAS. This suggests that the PAS-positive material in the IL-12 transgenic mice may have been material that was associated with mucin, a component of mucus. Second, IL-12 transgenic lungs also contained intraluminal cellular debris, resembling denuded airway epithelial cells. The sloughing of airway epithelial cells is likely secondary to the increased levels of oxidative stress, with generation of reactive oxygen species by the infiltrating macrophages. In keeping with these findings, Kay and colleagues (24) showed that increased oxidative stress enhances the loss of cilia in a rat tracheal culture system. Finally, cytokines such as IL-8 (1) and IL-13 (31) can directly inhibit the beat frequency of ciliated respiratory epithelial cells, a mechanism that can similarly be proposed for IL-12. Thus the combination of acellular and cellular debris and altered cytokine milieu in IL-12 transgenic airways contributed to delay the mucociliary clearance of the tracheobronchial tree, a finding also present in patients affected by Sjögren syndrome (35).

An increase in TGF-beta signaling in IL-12 transgenic lungs was reflected by increased expression of pSmad2, a cytoplasmic protein that becomes phosphorylated after ligation of the TGF-beta receptor 1. During TGF-beta signaling, pSmad2/Smad3 forms a complex with Smad4 that translocates into the nucleus and increases transcription of TGF-beta-responsive genes (34). TGF-beta is considered an immunosuppressive cytokine, involved in lymphocyte homeostasis and self-tolerance (6). In the absence of TGF-beta1, mice develop multiorgan inflammation/autoimmunity and die ~20 days after birth because of organ failure (49). Removal of B cells, CD4+ T cells, or CD8+ T cells alone does not improve longevity or inflammation, whereas removal of all T cells does improve both (7). Induction of TGF-beta in our autoimmune-type IL-12 transgenic model may have occurred as a compensatory mechanism, in response to the increased inflammation and oxidative stress, as a mechanism to suppress the autoimmune phenotype. Supporting this notion is a study that found that lupus-prone MRLlpr/lpr mice had decreased inflammation, decreased total IgG, and increased survival when given monthly intramuscular injections of a TGF-beta cDNA expression vector (46). Another study using mice that develop Sjögren-like salivary gland infiltrates found that calorie restriction in the older mice decreased salivary gland inflammation and that this correlated with an increase in TGF-beta and a decrease in inflammatory cytokines (9). Alternatively, the increase in pSmad2 in our model may reflect the increased inflammatory state of the IL-12 transgenic lung. In fact, the lungs of these mice have an increased number of infiltrating macrophages, which are known to produce TGF-beta. The increased TGF-beta signaling under conditions of inflammation can induce extracellular matrix protein production and lead to impaired healing and fibrosis (8).

The derangement of the normal lung NK cell population in this mouse model may provide additional insights into the pathogenic mechanisms underlying Sjögren syndrome. NK cells are a subset of lymphocytes characterized morphologically by large cytoplasmic granules and genetically by the lack of rearrangement in the gene segments forming B-cell or T-cell receptors. NK cells have been typically associated with innate immune responses, controlling intracellular pathogens (viruses, bacteria, and parasites) and tumors (21), but more recently they have also been implicated in adaptive immune responses (14). NK cells can in fact kill dendritic cells, limiting their antigen presentation capacity and overt immune reactions (16, 44).

In our model, NK cells showed a bimodal kinetics. They first significantly increased during the first month of life; then, between days 30 and 45, they dropped precipitously, remaining significantly lower than controls thereafter. Interestingly, the decline in NK cells preceded the appearance of the lung histological and functional lesions, suggesting a possible causal relationship. The initial increase in NK cells is in line with the well-established effects of IL-12. IL-12 was in fact first identified as a factor capable of enhancing NK cell activity and named "NK cell stimulatory factor" (28). The subsequent decrease in NK cells can also be explained as a direct effect of IL-12. It is known that prolonged exposure of lymphocytes (T, B, and NK) to IL-12 decreases their number and activity. For example, prolonged IL-12 treatment decreases the number of NK cells in the lungs of mice infected with Mycobacterium tuberculosis (40). Other factors, such as the observed increase in TGF-beta signaling, may contribute to the decline in NK cells. TGF-beta is a potent inhibitor of NK cell functions, capable of attenuating both NK cytolytic activity and IFN-{gamma} production (47).

Little is known about NK cells in Sjögren syndrome, but most studies have shown that in peripheral blood of Sjögren patients the number and/or activity of NK cells are decreased. Minato et al. (36) studied 13 patients with primary Sjögren syndrome and found a significantly depressed NK activity against measles-infected HeLa cells. Similar findings were reported by Miyasaka et al. (37) in 27 patients, by Pedersen et al. (43) in 21 patients, and by Struyf et al. (50) in 16 patients. Our mouse model provides evidence that a decrease in NK cells is associated with a Sjögren phenotype. In addition, the model shows that disease pathogenesis is initiated by prolonged exposure to increased IL-12 concentrations.

Together, these data suggest that IL-12 is necessary for the development of autoimmune pathology in specific organs. These organs include the lungs and salivary and lacrimal glands, organs that are specifically targeted in Sjögren disease. Our mouse model of IL-12 overexpression is novel and is the first to directly link overproduction of the Th1 cytokine IL-12 with autoimmune changes in the lung. This IL-12 transgenic model is also important in that it demonstrates several histological and functional characteristics that are found in Sjögren lung disease. Furthermore, the lung phenotype in this model progresses with age, which could allow for future testing of early interventions that might be useful in preventing, or halting, the progression of Sjögren disease.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institutes of Health (NIH) Grant DK-55670 and by NIH Grant CA-92871, which funds the Johns Hopkins Small Animal Imaging Resource Program.


   ACKNOWLEDGMENTS
 
The authors acknowledge Drs. Fred Askin and Grover Hutchins for their insights into lung histopathology, Dr. William Baldwin for revising the manuscript and initiating the multidisciplinary collaboration, and James Fox for assistance with X-Spect imaging.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Caturegli, Johns Hopkins Medical Institutions, Dept. of Pathology, Ross Bldg., Rm. 656, 720 Rutland Ave., Baltimore, MD 21205 (e-mail: pcat{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* S. McGrath-Morrow and B. Laube contributed equally to this work. Back


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 REFERENCES
 

  1. Allen-Gipson DS, Romberger DJ, Forget MA, May KL, Sisson JH, and Wyatt TA. IL-8 inhibits isoproterenol-stimulated ciliary beat frequency in bovine bronchial epithelial cells. J Aerosol Med 17: 107–115, 2004.[CrossRef][ISI][Medline]
  2. Aoki CA, Borchers AT, Li M, Flavell RA, Bowlus CL, Ansari AA, and Gershwin ME. Transforming growth factor beta (TGF-beta) and autoimmunity. Autoimmun Rev 4: 450–459, 2005.[CrossRef][ISI][Medline]
  3. Bacon CM, McVicar DW, Ortaldo JR, Rees RC, O'Shea JJ, and Johnston JA. Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL-12. J Exp Med 181: 399–404, 1995.[Abstract/Free Full Text]
  4. Battista G, Zompatori M, Poletti V, and Canini R. Thoracic manifestations of the less common collagen diseases. A pictorial essay. Radiol Med (Torino) 106: 445–453, 2003.[Medline]
  5. Birring SS, Patel RB, Parker D, McKenna S, Hargadon B, Monteiro WR, Falconer Smith JF, and Pavord ID. Airway function and markers of airway inflammation in patients with treated hypothyroidism. Thorax 60: 249–253, 2005.[Abstract/Free Full Text]
  6. Bommireddy R and Doetschman T. TGF-beta, T-cell tolerance and anti-CD3 therapy. Trends Mol Med 10: 3–9, 2004.[CrossRef][ISI][Medline]
  7. Bommireddy R, Engle SJ, Ormsby I, Boivin GP, Babcock GF, and Doetschman T. Elimination of both CD4+ and CD8+ T cells but not B cells eliminates inflammation and prolongs the survival of TGFbeta1-deficient mice. Cell Immunol 232: 96–104, 2004.[CrossRef][ISI][Medline]
  8. Branton MH and Kopp JB. TGF-beta and fibrosis. Microbes Infect 1: 1349–1365, 1999.[CrossRef][ISI][Medline]
  9. Chandrasekar B, McGuff HS, Aufdermorte TB, Troyer DA, Talal N, and Fernandes G. Effects of calorie restriction on transforming growth factor beta 1 and proinflammatory cytokines in murine Sjogren's syndrome. Clin Immunol Immunopathol 76: 291–296, 1995.[CrossRef][ISI][Medline]
  10. Chong MM, Metcalf D, Jamieson E, Alexander WS, and Kay TW. Suppressor of cytokine signaling-1 in T cells and macrophages is critical for preventing lethal inflammation. Blood 106: 1668–1675, 2005.[Abstract/Free Full Text]
  11. Constantopoulos SH, Papadimitriou CS, and Moutsopoulos HM. Respiratory manifestations in primary Sjogren's syndrome. A clinical, functional, and histologic study. Chest 88: 226–229, 1985.[CrossRef][ISI][Medline]
  12. Constantopoulos SH, Tsianos EV, and Moutsopoulos HM. Pulmonary and gastrointestinal manifestations of Sjogren's syndrome. Rheum Dis Clin North Am 18: 617–635, 1992.[ISI][Medline]
  13. Davidson BK, Kelly CA, and Griffiths ID. Ten year follow up of pulmonary function in patients with primary Sjogren's syndrome. Ann Rheum Dis 59: 709–712, 2000.[Abstract/Free Full Text]
  14. Degli-Esposti MA and Smyth MJ. Close encounters of different kinds: dendritic cells and NK cells take centre stage. Nat Rev Immunol 5: 112–124, 2005.[CrossRef][ISI][Medline]
  15. Eyles JL, Metcalf D, Grusby MJ, Hilton DJ, and Starr R. Negative regulation of interleukin-12 signaling by suppressor of cytokine signaling-1. J Biol Chem 277: 43735–43740, 2002.[Abstract/Free Full Text]
  16. Ferlazzo G, Tsang ML, Moretta L, Melioli G, Steinman RM, and Munz C. Human dendritic cells activate resting natural killer (NK) cells and are recognized via the NKp30 receptor by activated NK cells. J Exp Med 195: 343–351, 2002.[Abstract/Free Full Text]
  17. Foster WM, Walters DM, Longphre M, Macri K, and Miller LM. Methodology for the measurement of mucociliary function in the mouse by scintigraphy. J Appl Physiol 90: 1111–1117, 2001.[Abstract/Free Full Text]
  18. Fox RI. Sjogren's syndrome. Lancet 366: 321–331, 2005.[CrossRef][ISI][Medline]
  19. Groom J, Kalled SL, Cutler AH, Olson C, Woodcock SA, Schneider P, Tschopp J, Cachero TG, Batten M, Wheway J, Mauri D, Cavill D, Gordon TP, Mackay CR, and Mackay F. Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjogren's syndrome. J Clin Invest 109: 59–68, 2002.[CrossRef][ISI][Medline]
  20. Gudbjornsson B, Hedenstrom H, Stalenheim G, and Hallgren R. Bronchial hyperresponsiveness to methacholine in patients with primary Sjogren's syndrome. Ann Rheum Dis 50: 36–40, 1991.[Abstract/Free Full Text]
  21. Hamerman JA, Ogasawara K, and Lanier LL. NK cells in innate immunity. Curr Opin Immunol 17: 29–35, 2005.[CrossRef][ISI][Medline]
  22. Hasan M, Najjam S, Gordon MY, Gibbs RV, and Rider CC. IL-12 is a heparin-binding cytokine. J Immunol 162: 1064–1070, 1999.[Abstract/Free Full Text]
  23. Jamieson AM, Isnard P, Dorfman JR, Coles MC, and Raulet DH. Turnover and proliferation of NK cells in steady state and lymphopenic conditions. J Immunol 172: 864–870, 2004.[Abstract/Free Full Text]
  24. Kay RJ, Moate RM, Bray I, Sneyd JR, and Langton JA. Cultured rat trachea as a model for the study of ciliary abundance: the effect of oxygen. Anesthesiology 97: 275–277, 2002.[CrossRef][ISI][Medline]
  25. Kikawada E, Lenda DM, and Kelley VR. IL-12 deficiency in MRL-Faslpr mice delays nephritis and intrarenal IFN-{gamma} expression, and diminishes systemic pathology. J Immunol 170: 3915–3925, 2003.[Abstract/Free Full Text]
  26. Kimura H, Tzou SC, Rocchi R, Kimura M, Suzuki K, Parlow AF, Rose NR, and Caturegli P. Interleukin (IL)-12-driven primary hypothyroidism: the contrasting roles of two Th1 cytokines (IL-12 and interferon-gamma). Endocrinology 146: 3642–3651, 2005.[Abstract/Free Full Text]
  27. Kimura-Shimmyo A, Kashiwamura S, Ueda H, Ikeda T, Kanno S, Akira S, Nakanishi K, Mimura O, and Okamura H. Cytokine-induced injury of the lacrimal and salivary glands. J Immunother 25, Suppl 1: S42–S51, 2002.
  28. Kobayashi M, Fitz L, Ryan M, Hewick RM, Clark SC, Chan S, Loudon R, Sherman F, Perussia B, and Trinchieri G. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 170: 827–845, 1989.[Abstract/Free Full Text]
  29. Kolkowski EC, Reth P, Pelusa F, Bosch J, Pujol-Borrell R, Coll J, and Jaraquemada D. Th1 predominance and perforin expression in minor salivary glands from patients with primary Sjogren's syndrome. J Autoimmun 13: 155–162, 1999.[CrossRef][ISI][Medline]
  30. Koyama M, Johkoh T, Honda O, Mihara N, Kozuka T, Tomiyama N, Hamada S, and Nakamura H. Pulmonary involvement in primary Sjogren's syndrome: spectrum of pulmonary abnormalities and computed tomography findings in 60 patients. J Thorac Imaging 16: 290–296, 2001.[CrossRef][ISI][Medline]
  31. Laoukili J, Perret E, Willems T, Minty A, Parthoens E, Houcine O, Coste A, Jorissen M, Marano F, Caput D, and Tournier F. IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells. J Clin Invest 108: 1817–1824, 2001.[CrossRef][ISI][Medline]
  32. Laube BL, Auci RM, Shields DE, Christiansen DH, Lucas MK, Fuchs HJ, and Rosenstein BJ. Effect of rhDNase on airflow obstruction and mucociliary clearance in cystic fibrosis. Am J Respir Crit Care Med 153: 752–760, 1996.[Abstract]
  33. Laube BL, Karmazyn Y, Orens JB, and Mogayzel PJ. Impaired mucociliary clearance following lung transplantation is improved by albuterol inhalation (Abstract). J Aerosol Med 26: 195, 2003.
  34. Li MO, Wan YY, Sanjabi S, Robertson AK, and Flavell RA. Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol 24: 99–146, 2006.[CrossRef][ISI][Medline]
  35. Mathieu A, Cauli A, Pala R, Satta L, Nurchis P, Loi GL, Garau P, Sanna G, Passiu G, and Carta P. Tracheo-bronchial mucociliary clearance in patients with primary and secondary Sjogren's syndrome. Scand J Rheumatol 24: 300–304, 1995.[ISI][Medline]
  36. Minato N, Takeda A, Kano S, and Takaku F. Studies of the function of natural killer-interferon system in patients with Sjogren syndrome. J Clin Invest 69: 581–588, 1982.[ISI][Medline]
  37. Miyasaka N, Seaman W, Bakshi A, Sauvezie B, Strand V, Pope R, and Talal N. Natural killing activity in Sjogren's syndrome. An analysis of defective mechanisms. Arthritis Rheum 26: 954–960, 1983.[ISI][Medline]
  38. Mustafa W, Zhu J, Deng G, Diab A, Link H, Frithiof L, and Klinge B. Augmented levels of macrophage and Th1 cell-related cytokine mRNA in submandibular glands of MRL/lpr mice with autoimmune sialoadenitis. Clin Exp Immunol 112: 389–396, 1998.[CrossRef][ISI][Medline]
  39. Newball HH and Brahim SA. Chronic obstructive airway disease in patients with Sjogren's syndrome. Am Rev Respir Dis 115: 295–304, 1977.[ISI][Medline]
  40. Nolt D and Flynn JL. Interleukin-12 therapy reduces the number of immune cells and pathology in lungs of mice infected with Mycobacterium tuberculosis. Infect Immun 72: 2976–2988, 2004.[Abstract/Free Full Text]
  41. Ohyama Y, Nakamura S, Matsuzaki G, Shinohara M, Hiroki A, Fujimura T, Yamada A, Itoh K, and Nomoto K. Cytokine messenger RNA expression in the labial salivary glands of patients with Sjogren's syndrome. Arthritis Rheum 39: 1376–1384, 1996.[ISI][Medline]
  42. Papiris SA, Maniati M, Constantopoulos SH, Roussos C, Moutsopoulos HM, and Skopouli FN. Lung involvement in primary Sjogren's syndrome is mainly related to the small airway disease. Ann Rheum Dis 58: 61–64, 1999.[Abstract/Free Full Text]
  43. Pedersen BK, Oxholm P, Manthorpe R, and Andersen V. Interleukin 2 augmentation of the defective natural killer cell activity in patients with primary Sjogren's syndrome. Clin Exp Immunol 63: 1–7, 1986.[ISI][Medline]
  44. Piccioli D, Sbrana S, Melandri E, and Valiante NM. Contact-dependent stimulation and inhibition of dendritic cells by natural killer cells. J Exp Med 195: 335–341, 2002.[Abstract/Free Full Text]
  45. Potena A, La Corte R, Fabbri LM, Papi A, Trotta F, and Ciaccia A. Increased bronchial responsiveness in primary and secondary Sjogren's syndrome. Eur Respir J 3: 548–553, 1990.[Abstract]
  46. Raz E, Dudler J, Lotz M, Baird SM, Berry CC, Eisenberg RA, and Carson DA. Modulation of disease activity in murine systemic lupus erythematosus by cytokine gene delivery. Lupus 4: 286–292, 1995.[Abstract/Free Full Text]
  47. Rook AH, Kehrl JH, Wakefield LM, Roberts AB, Sporn MB, Burlington DB, Lane HC, and Fauci AS. Effects of transforming growth factor beta on the functions of natural killer cells: depressed cytolytic activity and blunting of interferon responsiveness. J Immunol 136: 3916–3920, 1986.[Abstract]
  48. Santiago-Raber ML, Laporte C, Reininger L, and Izui S. Genetic basis of murine lupus. Autoimmun Rev 3: 33–39, 2004.[CrossRef][ISI][Medline]
  49. Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel G, Calvin D, Annunziata N and Doetschman T. Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature 359: 693–699, 1992.[CrossRef][Medline]
  50. Struyf NJ, Snoeck HW, Bridts CH, De Clerck LS, and Stevens WJ. Natural killer cell activity in Sjogren's syndrome and systemic lupus erythematosus: stimulation with interferons and interleukin-2 and correlation with immune complexes. Ann Rheum Dis 49: 690–693, 1990.[Abstract/Free Full Text]
  51. Taouli B, Brauner MW, Mourey I, Lemouchi D, and Grenier PA. Thin-section chest CT findings of primary Sjogren's syndrome: correlation with pulmonary function. Eur Radiol 12: 1504–1511, 2002.[CrossRef][ISI][Medline]
  52. Tesch GH, Maifert S, Schwarting A, Rollins BJ, and Kelley VR. Monocyte chemoattractant protein 1-dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Faslpr mice. J Exp Med 190: 1813–1824, 1999.[Abstract/Free Full Text]
  53. Venables PJ. Sjogren's syndrome. Best Pract Res Clin Rheumatol 18: 313–329, 2004.[Medline]
  54. Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE, Daniels TE, Fox PC, Fox RI, Kassan SS, Pillemer SR, Talal N, and Weisman MH. Classification criteria for Sjogren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 61: 554–558, 2002.[Abstract/Free Full Text]
  55. Wilson WR and Bedell GN. The pulmonary abnormalities in myxedema. J Clin Invest 39: 42–55, 1960.[ISI][Medline]
  56. Wright SA, Convery RP, and Liggett N. Pulmonary involvement in Sjogren's syndrome. Rheumatology (Oxford) 42: 697–698, 2003.[Medline]



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