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The Journal of Immunology, 2007, 179, 2318-2329
Copyright © 2007 by The American Association of Immunologists, Inc.
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Development of Sjögren’s Syndrome in Nonobese Diabetic-Derived Autoimmune-Prone C57BL/6.NOD-Aec1Aec2 Mice Is Dependent on Complement Component-31

Cuong Q. Nguyen2,*, Hyuna Kim*, Janet G. Cornelius{dagger} and Ammon B. Peck*,{dagger},{ddagger}

* Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL 32610; {dagger} Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32610; and {ddagger} Center for Orphan Autoimmune Diseases, College of Dentistry, University of Florida, Gainesville, FL 32610


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of complement in the etiology of Sjögren’s syndrome (SjS), a human autoimmune disease manifested primarily by salivary and lacrimal gland dysfunction resulting in dry mouth/dry eye syndrome, remains ill-defined. In the present study, we examined the role of complement component-3 (C3) using a newly constructed C3-gene knockout mouse, C57BL/6.NOD-Aec1Aec2.C3–/–. Inactivation of C3 in the parental C57BL/6.NOD-Aec1Aec2 strain, a model of primary SjS, resulted in a diminished or total absence of both preclinical and clinical manifestations during development and onset of disease, including reduced acinar cell apoptosis, reduced levels of caspase-3, lack of leukocyte infiltration of submandibular glands, reduced synthesis of disease-associated autoantibodies, maintenance of normal glandular architecture, and retention of normal saliva secretion. In addition, C57BL/6-NOD.Aec1Aec2.C3–/– mice did not exhibit increased numbers of marginal zone B cells, a feature of SjS-prone C57BL/6-NOD.Aec1Aec2 mice. Interestingly, C57BL/6-NOD.Aec1Aec2.C3–/– mice retained some early pathological manifestations, including activation of serine kinases with proteolytic activity for parotid secretory protein. This improvement in the clinical manifestations of SjS-like disease in C57BL/6.NOD-Aec1Aec2.C3–/– mice, apparently a direct consequence of C3 deficiency, supports a much more important role for complement in the adaptive autoimmune response than previously recognized, possibly implicating an essential role for innate immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sjögren’s syndrome (SjS)3 is a human autoimmune disease characterized by loss of exocrine function as a result of a chronic immune attack directed primarily against the salivary and lacrimal glands leading to xerostomia sicca (dry mouth) and keratoconjunctivitis sicca (dry eyes), respectively (1, 2, 3, 4). Onset of clinical symptoms of SjS is highly dependent on the activity of B lymphocytes, including the well-documented hyperreactivity of B cells, extreme hypergammaglobulinemia, production of numerous autoantibodies, and eventually glandular dysfunction (1, 2, 5, 6, 7, 8, 9, 10, 11, 12). One mechanism known to control survival, activation, and proliferation of B cells is through cross-linking of their Ag receptors (BCR) and their coreceptors, especially CD19 and CD21. Cross-linking of BCR and coreceptors resulting in a hyperproliferation of B cells involves C3d (13). Although the ligand for CD19 remains unknown, CD19 can form a noncovalent quaternary complex that involves CD21, CD81, and CD225 (14). CD21, in turn, is a receptor for C3d, a proteolytic product of complement component-3 (C3) that forms covalent bonds with foreign Ag or immune complexes. Signals generated by coligations of CD19/CD21 and BCR via C3d fragments act as positive regulators of B cell activation, thereby lowering the threshold for B cell activation by Ag. In this way, the C3d/Ag complex(es) may contribute to the hyperproliferation and hyperreactivity of autoreactive B cells (15), a common feature of SjS.

Although the underlying cause of SjS remains elusive, studies using various mouse models of SjS have begun to provide new insights into the cellular and molecular mechanisms important to understanding this disease (16). One of the better models is the NOD mouse, which uniquely exhibits salivary and lacrimal gland dysfunction concomitant with appearance of leukocyte infiltrations of the exocrine glands (17), together with its many congenic strains with known genetic differences (18, 19, 20, 21, 22). In a recent study using NOD.B10-H2b mice, a model of primary SjS (23), we investigated the possible role of C3 in the development and onset of SjS-like disease by treating these mice with cobra venom factor (CVF) (24), a substance known to deplete circulating C3. Treatment of NOD.B10-H2b mice starting at time of onset of disease, while not preventing the aberrant pathophysiological activities defining the preclinical disease, actually reduced the severity of leukocyte infiltrations into the salivary and lacrimal glands, the production of autoantibodies, as well as the degree of exocrine gland dysfunction, all defining the onset of clinical disease. Because NOD mice are deficient in C5, involvement of the membrane attack complex seems unlikely. In contrast, this reduction in clinical disease severity correlated with significant reductions in the levels of CD19/CD21 coexpression on B cells, while no changes were noted in expression levels of CD22 on CD19-positive B cells (24).

These initial findings suggested a direct correlation between C3 depletion, loss of CD19high/CD21high B cell subpopulations, and reduced autoimmunity in CVF-treated NOD.B10-H2b mice (24). However, the use of CVF to deplete C3 may not have induced these changes directly, but may have influenced the disease indirectly, e.g., by the known presence of impurities in CVF and/or by a deviation of immune reactivity due to the strong immunogenicity of CVF per se (25). In addition, the impact of complement on the early phases of SjS, specifically the development of and/or the innate immunity underlying SjS has never been completely investigated. For this reason, we have re-examined the role of C3 in the development and onset of SjS-like disease using a newly constructed C3 gene knockout (KO) mouse, C57BL/6.NOD-Aec1Aec2.C3–/–. We present here results showing that C57BL/6.NOD-Aec1Aec2 mice carrying a disrupted C3 gene failed to develop an autoimmune response against the salivary glands, thereby exhibiting no clinical SjS-like disease. Unexpectedly, these mice also failed to exhibit many of the pathophysiological attributes of the preclinical stage of SjS-like disease suggesting a more complex role for C3 in establishing an autoimmune environment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

All mice used in this study were bred and maintained under specific pathogen-free conditions in the Department of Animal Care Services within the Health Science Center (University of Florida, Gainesville, FL). To construct the C3 gene KO mouse, male B6.129S4-C3tm1Crr/J mice purchased from The Jackson Laboratory were mated to female C57BL/6.NOD-Aec1Aec2 mice, whose derivation is presented elsewhere (18, 21). F1 heterozygotes were intercrossed to produce an F2 generation that was screened for homozygosity at the Aec1 locus, Aec2 locus, and the C3 gene. The Aec1 and Aec2 loci were identified by microsatellite marker (Dmit) genotyping, as described elsewhere (18). PCR primers for C3 and the neomycin-disrupted C3 gene were purchased from Invitrogen Life Technologies. Primers for the Dmit markers were based on sequences obtained from The Jackson Laboratory. One male and one female, shown to be homozygous for the Aec1 and Aec2 loci, as well as the disrupted C3 gene, were bred to establish the C57BL/6.NOD-Aec1Aec2.C3–/– line. This line was carried via a single line descent through brother-sister mating. All mice received water and food ad libitum. Studies described herein were approved by the University of Florida Institutional Animal Care and Use Committee.

Measurement of stimulated saliva flow rates

To measure stimulated flow rates of saliva, individual mice were weighed and given an i.p. injection of 100 µl of a mixture containing isopreterenol (0.2 mg/1 ml of PBS) and pilocarpine (0.05 mg/1 ml of PBS). Saliva was collected for 10 min from the oral cavity of individual mice using a micropipette starting 1 min after injection of the secretagogue. The volume of each saliva sample was measured. The saliva samples were then frozen at –80°C until analyzed.

Detection of proteolytic activity for parotid secretory protein (PSP)

Detection of PSP proteolysis was conducted by incubating whole saliva specimens with a synthesized oligopeptide corresponding to amino acids 20 through 34 of the published sequence for mouse PSP. This oligopeptide contains the proteolytic site (NLNL) for a serine kinase present in salivary glands during development and onset of SjS-like disease in the NOD mouse (C. Q. Nguyen, V. Brown, and A. B. Peck, our unpublished data). Eight microliters of saliva collected from individual mice was mixed with 42 µl of the PSP oligopeptide (2.5 mg/ml) and incubated at 42°C for 12 h. Following incubation, 50 µl Tris-HCl buffer (50 mM (pH 8.0)) was added and the mixture centrifuged through Microspin filter tubes at 14,000 rpm for 10 min. The filtrates were analyzed by HPLC (Dionex Systems) for proteolytic products. Control samples consisted of 50 µl of the PSP oligopeptide.

Histology

Submandibular glands were surgically removed from each mouse at time of euthanasia, and placed in 10% phosphate-buffered formalin for 24 h. Fixed tissues were embedded in paraffin and sectioned at 5 µm thickness. Paraffin-embedded slides were deparaffinized by immersing in xylene, followed by dehydrating in ethanol. The tissue sections were stained with H&E dye (Gainesville Service Tech). Stained sections were observed at x100 magnification for glandular structure and leukocyte infiltration. The number of lymphocytic focus per submandibular section was counted in a blinded fashion by two investigators.

Immunofluorescent staining for B and T lymphocytes

Paraffin-embedded tissues of the submandibular glands were sectioned and mounted onto microscope slides. Slides were deparaffinized by immersing in xylene, then dehydrated in ethanol. Following a 5-min wash with PBS at 25°C, the sections were incubated 1 h with blocking solution containing normal rabbit serum diluted 1/50 in PBS. Each section was incubated with rat anti-mouse B220 (BD Biosciences/BD Pharmingen) diluted 1/10 and goat anti-mouse CD3 (Santa Cruz Biotechnology) diluted 1/50 for 1 h at 25°C. The slides were washed three times with PBS for 5 min per wash followed by a 1-h incubation with Texas Red-conjugated rabbit anti-rat IgG (Biomeda) diluted 1/25 and FITC-conjugated rabbit anti-goat IgG (Sigma-Aldrich) diluted 1/100 at 25°C. The slides were washed thoroughly with PBS, treated with Vectashield 4',6'-diamidino-2-phenylindole (DAPI)-mounting medium (Vector Laboratories) and overlaid with glass coverslips. Stained sections were visualized at x200 magnification.

Flow cytometry for subpopulations of B cells

Spleens were freshly explanted from euthanized mice and gently minced through a steel sieve. Following a single wash with PBS, RBC were lysed by 7-min incubation in 0.84% NH4Cl. The resulting cell suspensions were washed two times in PBS, counted, and resuspended in FACS buffer (PBS supplemented to 2% BSA and 0.01% NaN3) to 1 x 108 cells/ml. Aliquots of each cell preparation containing 1 x 105 cells were incubated 45 min with either R-PE-conjugated rat anti-mouse CD19 mAb (no. 557399), FITC-conjugated rat anti-mouse CD19 mAb (no. 557398), FITC-conjugated rat anti-mouse CD21 mAb (no. 553818), or PE-conjugated goat anti-mouse CD23 (no. 553139; BD Biosciences/BD Pharmingen), washed in FACS buffer, then analyzed for fluorescence staining on a FACScan (BD Biosciences).

Detection of cleaved products of caspase-3 by immunohistochemistry

Following euthanasia of the mice, their submandibular glands were surgically removed at the ages designated in the text, placed in 10% phosphate-buffered formalin for 24 h, then embedded in paraffin and sectioned at 5 µm thickness. Paraffin-embedded slides were deparaffinized by immersing in xylene, followed by dehydrating in ethanol. The tissue sections were washed in PBS for 5 min, and then incubated 15 min at 25°C in Sniper blocking solution (BT967H; BioCare Medical). Each section was incubated with rabbit anti-cleaved caspase-3 diluted at 1/400 (CP229B; BioCare Medical) overnight at 25°C. The slides were washed three times with PBS for 5 min per wash, followed by 30-min incubation at 25°C with Mach-2 goat anti-rabbit HRP polymer secondary Ab (RHRP520; BioCare Medical). The slides were washed again with PBS, stained with Cardassian diaminobenzidine chromagen (DBC859L10; Biocare Medical), rinsed in deionized water, and counterstained with methyl green (S1962; DakoCytomation). Stained sections were visualized at x200 magnification. The number of caspase-3-positive cells per submandibular section was counted in a blinded fashion by two different individuals.

Measurement of salivary protein concentrations and detection of amylase activity

Total protein content was determined using the Bradford method. Amylase activity in saliva was determined using the Infinity Liquid Amylase kit (Thermo Trace Electron) in which starch was the substrate. Saliva samples were diluted 250-fold with deionized water and added to 1 ml of the Infinity Amylase Liquid Stable Reagent. Following 1- and 2-min incubators at 37°C, absorbance was measured at a wavelength of 405 nm. Amylase activity was calculated according to the manufacturer’s instructions using the formula: amylase activity (U/L) = {Delta} A/2 x 5140 x 400 (sample dilution).

Detection of anti-nuclear autoantibody (ANA) patterns

ANAs in the sera of mice were detected using an ANA screening kit (Immunoconcepts). Sera were tested at 1/40, 1/100, 1/500, and 1/1000 dilutions. Presented in this study, however, are data from using the sera at 1/40 dilutions. In brief, HEp-2 fixed substrate slides were overlaid with the appropriate mouse serum. Slides were incubated for 30 min at room temperature in a humidified chamber. After three washes for 5 min with PBS, the substrate slides were covered with FITC-conjugated goat anti-mouse IgG (Sigma-Aldrich) diluted 1/50 for 30 min at room temperature. After three washes, nuclear fluorescence was detected by fluorescence microscopy at x200 magnification.

Quantification of salivary pro-matrix metalloproteinase (MMP)-9 levels

Pro-MMP-9 levels in saliva samples were qualified using the mouse pro-MMP-9 ELISA kit (DY909; R&D Systems). In brief, capture Ab was coated onto 96-well microtiter plates overnight at 4°C. Saliva samples diluted 50% were added to wells and incubated for 2 h at room temperature. After washing with wash buffer (PBS, 0.05% Tween 20), detection Ab was added to each well, followed by addition of streptavidin-HRP. Each sample was run in duplicate. The standard curve was generated using recombinant mouse pro-MMP diluted over the range from 0 to 40 ng/ml. Color was developed with tetramethylbenzidine (Sigma-Aldrich) for ~30 min and stopped by adding 50 µl of 2 N H2SO4. OD readings were determined at 450 nm with wavelength correction at 590 nm for the background.

Detection of Ig-specific muscarinic acetylcholine type 3 receptor (M3R) autoantibodies

Detection of anti-M3R Abs in sera of C57BL/6.NOD-Aec1Aec2 mice was determined as described in detail elsewhere (26). In brief, Flp-In CHO cells transfected with mM3R were collected from growing cultures, washed once with PBS, and resuspended in FACS buffer (PBS, 0.5% BSA, 0.07% NaN3). Aliquots of cells at a density of 1 x 106 cells/0.1 ml were incubated 2 h at 4°C with 10 µl of sera from individual mice or pooled from appropriate groups. Cells were washed once with FACS buffer, resuspended in 50 µl of FACS buffer, and incubated for 30 min at 4°C with either FITC-conjugated goat anti-mouse IgG1, IgG2b, IgG2c, IgG3, IgM, IgA, IgE, and IgD (Southern Biotechnology Associates). After a final wash with FACS buffer, the cells were resuspended in FACS buffer and analyzed using a FACScan cytometer equipped with Cell Quest software (BD Biosciences). Control reactions included transfected cells incubated with secondary Ab alone (shown herein), appropriate isotype controls which exhibited profiles similar to secondary Ab alone (data not shown) and test reactions on nontransfected Flp-In CHO cells (data not shown). An increase in fluorescence intensity compared with secondary Ab alone was considered a positive reaction.

Statistical analyses

All values presented represent the means ± SE. Statistical differences were analyzed with either the Student-Newman-Keuls or {chi}2 tests. Values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Pathophysiological characteristics of SjS-like disease in C57BL/6.NOD-Aec1Aec2.C3–/– mice

Although the underlying cause of SjS remains elusive, a number of studies using the NOD mouse model and its congenic partner strains have led us to propose the concept that this autoimmune exocrinopathy progresses in three continuous, consecutive, yet distinct phases (27, 28). In phase 1, a series of aberrant genetic, physiological, and biochemical activities associated with retarded salivary gland organogenesis and acinar cell apoptosis occur before and independent of initiation of an autoimmune attack. In phase 2, leukocytes infiltrate the exocrine glands, possibly as a result of glandular cell injury in phase 1, with a concomitant increase in the expression of inflammatory cytokines and production of autoantibodies. In phase 3, secretory dysfunction of the salivary and lacrimal glands occurs, most likely the result of production of Abs reactive with the muscarinic acetylcholine receptors (6, 29), marked by significant loss of secreted proteins. An aberration within any one of these three phases, e.g., disruption of either the IFN-{gamma} (20) or IL4 gene (19, 22), interrupts subsequent onset of clinical SjS-like disease.

To identify development of preclinical SjS-like disease in C57BL/6.NOD-Aec1Aec2 mice, we commonly test for the appearance in saliva (or submandibular gland tissue lysates) of the disease-associated activation of a serine kinase capable of proteolysis of PSP. Saliva samples collected from individual C57BL/6.NOD-Aec1Aec2 and C3 gene knockout C57BL/6.NOD-Aec1Aec2. C3–/– mice at various ages (4, 8, 12, 16, and 25 wk) were tested for their ability to clip a 16-mer surrogate oligopeptide of PSP at the targeted NLNL amino acid sequence present in the N terminus. As presented in Fig. 1, saliva samples from randomly selected C57BL/6.NOD-Aec1Aec2 and C57BL/6.NOD-Aec1Aec2.C3–/– mice exhibited proteolysis of PSP as early as 13 wk of age (see Table I), and continued out to at least 24 wk of age. As the actual levels of PSP proteolysis do not always correlate with severity of subsequent disease, a positive assay result is used primarily to determine the possibility for SjS-like disease development.


Figure 1
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  		FIGURE 1. Detection of aberrant serine proteolytic activity in saliva of C57BL/6.NOD-Aec1Aec2 and C57BL/6.NOD-Aec1Aec2.C3–/– mice, but not C57BL/6 mice. Saliva collected from individual animals between the ages of 4 and 24 wk were incubated with the synthetic oligopeptide EAVPQNLNLDVELLQQ representing a sequence from the N terminus of PSP with the NLNL proteolytic site for activated serine kinases. Following 2-h incubation at 42°C, each reaction was diluted with Tris-HCl, centrifuged through Microspin filter tubes, and the filtrates were separated by HPLC. Synthesized PSP oligopeptide, whether analyzed in buffer (A) or in the presence of control C57BL/6 saliva (B) reproducibly elutes at 13.8 min (A). PSP oligopeptide, when incubated with saliva from C57BL/6.NOD-Aec1Aec2 mice known to be predisposed to develop SjS-like disease and to contain activated serine kinases, is digested to produce the two cleaved fragments that elute at ~9.0 and 12.5 min (C). Proteolysis of the synthetic PSP oligopeptide was observed when incubated in saliva from C57BL/6.NOD-Aec1Aec2.C3–/– mice (D). Although saliva from each mouse in each of the three groups were tested for enzyme activity, the data presented depict a single, representative HPLC run from each group.

 

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  		Table I. Summary of various disease parameters defining SjS in C57BL/6.NOD-Aec1Aec2 versus C57BL/6 and C57BL/6.NOD-Aec1Aec2.C3–/– mice

 
Additional markers of physiological changes in acinar tissue that also appear before leukocyte infiltration into the salivary (and lacrimal glands) include an increase in the levels of cellular apoptosis, as determined by increased caspase-3 activity and/or TdT-TUNEL staining, MMP-9 levels as determined by ELISA, and Fas-FasL expression (30). To support the above observation with PSP, levels of acinar cell apoptosis was determined for the submandibular glands of C57BL/6.NOD-Aec1Aec2.C3–/– and C57BL/6.NOD-Aec1Aec2 mice compared with age- and sex-matched C57BL/6 mice. As presented in Fig. 2 and Table I, young (<8 wk of age) C57BL/6.NOD-Aec1Aec2 mice predisposed to SjS-like disease exhibited a significantly increased level of apoptosis (>4-fold; p < 0.001) compared with C57BL/6 mice, as measured by detection of cleavage products of caspase-3 in their submandibular glands. Interestingly, C57BL/6.NOD-Aec1Aec2.C3–/– mice showed no significant increase in the level of glandular apoptosis from 4 to 24 wk of age. TUNEL staining further confirmed the observation that elimination of C3 reversed the early wave of apoptosis to normal levels similar to C57BL/6 mice (data not shown).


Figure 2
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  		FIGURE 2. Temporal changes in levels of apoptosis in submandibular glands of C57BL/6.NOD-Aec1Aec2 and C57BL/6.NOD-Aec1Aec2.C3–/– mice using cleaved caspase-3 by immunohistochemistry. A, Detection of cleaved products of caspase-3 on histological sections of submandibular glands from C57BL/6 mice at 4 (a) and 27 (b) wk of age, C57BL/6.NOD-Aec1Aec2 mice at 4 (c) and 27 (d) wk of age, and C57BL/6.NOD-Aec1Aec2.C3–/– mice at 4 (e) and 27 (f) wk of age (n = 4 mice in each group and at each time point). B, Quantification of apoptotic events observed in the submandibular glands of C57BL/6 (black fill), C57BL/6.NOD-Aec1Aec2 (stippled fill), and C57BL/6.NOD-Aec1Aec2.C3–/– (clear fill) mice during the early (4–7 wk of age) vs late (24–27 wk of age) stages of disease. Statistical differences were analyzed by the Student-Newman-Keuls test using GraphPad InStat3. ***, p < 0.001; NS, not statistically significant.

 
The early phase of SjS also encompasses dynamic changes in the glandular physiology that suggests the glandular tissues play an active role in the initiation of the autoimmune process (16). The extracellular matrix (ECM) is thought to modulate cellular differentiation, tissue morphogenesis, glandular development, and organ function (31). Increased levels of ECM remodeling by MMPs has been implicated in the early phases of SjS development, with the most prominently expressed matrix degrading enzyme being MMP-9 (32). As presented in Table I, the absolute levels of saliva (pro-) MMP-9 increased from 4 to 24–27 wk of age in C57BL/6.NOD-Aec1Aec2 (p < 0.001) and C57BL/6.NOD-Aec1Aec2.C3–/– (p = NS) mice, contrary to C57BL/6 mice which showed constant levels of (pro-) MMP-9 over the same time period. Thus, these data suggest that C57BL/6.NOD-Aec1Aec2.C3–/– mice, similar to C57BL/6.NOD-Aec1Aec2 mice, retain the PSP proteolytic activity, the earliest marker of disease, and exhibited increased (although not statistically significant) pro-MMP-9 levels. However, the C3 KO mice did not show increased levels of acinar tissue apoptosis. With development of acinar cell dysfunction in C57BL/6.NOD-Aec1Aec2 mice, both saliva protein concentration and {alpha}-amylase (the most abundant protein in saliva) increase as saliva volumes decrease (Table I). In contrast, parental C57BL/6 mice, despite exhibiting a slight increase in their saliva protein concentrations, did not show an increase in {alpha}-amylase activity. C57BL/6.NOD-Aec1Aec2.C3–/– mice exhibited relatively stable protein concentrations as well as {alpha}-amylase activities (Table I). Together, these data suggest that a lack of C3 has selective impact on the preimmune pathophysiological changes in the submandibular glands, but most importantly reduces acinar cell death resulting subsequently in relatively normal physiological processes.

Altered autoimmune reactivity in C57BL/6.NOD-Aec1Aec2.C3–/– mice

Lymphocyte infiltration of the submandibular and/or lacrimal glands is a critical criterion for identification of the autoimmune phase of SjS in both human and animal models. Although the number of lymphocytic foci present in the salivary and lacrimal glands often does not often correlate directly with disease or its severity (22), SjS patients and NOD-derived mouse strains exhibiting SjS-like disease typically have lymphocytic infiltrates in their salivary glands, histologically termed lymphoepithelial sialadenitis. To determine whether C57BL/6.NOD-Aec1Aec2.C3–/– mice develop lymphoepithelial sialadenitis, the submandibular glands from both male and female mice euthanized at various ages between 4 and 36 wk were freshly explanted, fixed in formalin, embedded in paraffin, sectioned, and stained with H&E (Fig. 3). Histological examinations revealed that multiple foci of leukocytic infiltrates in submandibular glands were detected first at ~10 wk of age in C57BL/6.NOD-Aec1Aec2 (data not presented) and continued to increase in number and size with time. No leukocytic infiltrations were observed in C57BL/6 and C57BL/6.NOD-Aec1Aec2.C3–/– mice by 25 wk of age. However, by 36 wk of age, leukocytes could be detected in the submandibular glands of both C57BL/6 and C57BL/6.NOD-Aec1Aec2.C3–/– mice (Fig. 3, C and K, respectively). Quantitative comparison of the number of lymphocytic foci in C57BL/6.NOD-Aec1Aec2, C57BL/6.NOD-Aec1Aec2.C3–/–, and C57BL/6 mice over time is presented in Table II. These leukocytic infiltrates in the C57BL/6.NOD-Aec1Aec2.C3–/– mice are considered to be a consequence of the genetic background of C57BL/6 and not of the SjS predisposed phenotype of the C57BL/6.NOD-Aec1Aec2 parental mice.


Figure 3
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  		FIGURE 3. Histological examination of submandibular glands C57BL/6, C57BL/6.NOD-Aec1Aec2, and C57BL/6.NOD-Aec1Aec2.C3–/– mice at various ages. Submandibular glands were removed at time of euthanization from mice within each group, fixed in 10% formalin, embedded in paraffin, and cut for 5-µm thickness serial sections. Each section was stained with Mayer’s H&E dye (A–C, E–G, and I–K) to identify the presence or absence of leukocytic foci. Foci were present only in the glands C57BL/6.NOD-Aec1Aec2 mice before 30 wk of age (as depicted in F). After 30 wk, infiltrates were observed in all three strains (C, G, and K). Immunohistological staining for CD3+ T (green) and B220 B (red) cells showed the presence of each cell populations within the infiltrates (D, H, and L). Nuclei were stained with DAPI (blue).

 

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  		Table II. Quantification of lymphocytic foci in C57BL/6.NOD-Aec1Aec2versus C57BL/6 and C57BL/6.NOD-Aec1Aec2.C3–/– mice

 
Immunohistological staining revealed that the lymphocytic foci present in the submandibular glands of C57BL/6.NOD-Aec1Aec2 were comprised of distinct regions of CD3+ T and B220+ B cells localized in well-defined foci irrespective of the age examined (Fig. 3H). In contrast, leukocytic infiltrations observed at 33–36 wk of age in both parental C57BL/6 (Fig. 3D) and C57BL/6.NOD-Aec1Aec2.C3–/– (Fig. 3L) mice lacked a similar organization of T and B cells, appearing more sporadic within periductal infiltrates. Interestingly, the mature lymphocytic foci observed in the submandibular glands of C57BL/6.NOD-Aec1Aec2 mice were comprised predominantly of B cells, thus correlating with the hyperproliferation and expansion, as well as the dependency of SjS-like disease on B cells. To determine whether differences exist in the B cell populations between C57BL/6.NOD-Aec1Aec2, C57BL/6.NOD-Aec1Aec2.C3–/–, and parental C57BL/6 mice during the development and onset of SjS-like disease, the phenotypes of the B cell populations present in the spleens at the time of disease onset in the submandibular glands were examined. Splenic CD19-positive B cells from each strain were isolated and analyzed by flow cytometry, identifying marginal zone (MZ) B cells and follicular (FO) B cells based on the differential expression of CD21 and CD23 surface molecules. Previously, we found that treatment of mice with CVF resulted in a 2-fold decrease in the expression levels of CD21 and CD23 on the surface of splenic B cells when compared with PBS-treated animals (24). As presented in Fig. 4, there was nearly a 2-fold increase in the percentage of MZ B cells in C57BL/6.NOD-Aec1Aec2 mice compared with either C57BL/6 or C57BL/6.NOD-Aec1Aec2.C3–/– mice. In contrast, only the female mice of the C57BL/6.NOD-Aec1Aec2 line and the male mice of the C57BL/6.NOD-Aec1Aec2.C3–/– line showed a decrease in FO B cells. Although the underlying reasons for these differences between male vs female mice are not known, they may reflect the different disease phenotypes exhibited by male and female mice, namely, that males have a more severe lacrimal gland disease while females have a more severe salivary gland disease (16, 23, 28).


Figure 4
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  		FIGURE 4. Identification of MZ vs FO B cell populations in the spleens of female and male C57BL/6, C57BL/6.NOD-Aec1Aec2, and C57BL/6.NOD-Aec1Aec2.C3–/– mice. Single-cell suspensions of splenic leukocytes from 24-wk-old C57BL/6 (n = 3, left panels), 26-wk-old C57BL/6.NOD-Aec1Aec2 (n = 3, middle panels), or 27-wk-old C57BL/6.NOD-Aec1Aec2.C3–/– (n = 4, right panels) female and male mice were incubated with FITC-conjugated rat anti-mouse CD21 mAb and R-PE-conjugated rat anti-mouse CD23, then analyzed by flow cytometry for differential fluorescence intensity based on CD21 and CD23 markers to identify MZ (CD21highCD23int), and FO (CD21intCD23low) B cells. Data presented in A and B are representative flow cytometric analysis of MZ and FO B cells in individual mice, while the cumulative data from each experimental group of female and male mice are presented in C and D, respectively. Data presented are the means ± SE. Statistical differences were analyzed with the Student-Newman-Keuls test using GraphPad InStat3. **, p < 0.01; ***, p < 0.001.

 
With the appearance of T and B lymphocytes within the submandibular glands of C57BL/6.NOD-Aec1Aec2 mice, plus the detectable changes within their splenic B cell populations, an increasing number of serum autoantibodies can be detected (33, 34, 35, 36). Of particular importance in human SjS disease (but not in SjS-like disease of NOD mice) are the ANAs, especially anti-SS-A/Ro and anti-SS-B/La, as the presence of these ANAs in the sera of human SjS patients is one of the diagnostic markers for clinical disease (4). To identify the general presence of ANAs in the sera of C57BL/6.NOD-Aec1Aec2.C3–/– mice, individual animals were serially bled between 4 and 22 wk of age and their sera were prepared. Each serum was then tested individually for reactivity on HEp-2 cells and visualized by staining with FITC-conjugated goat anti-mouse whole IgG. As presented in Fig. 5, the frequency (percent) of C57BL/6.NOD-Aec1Aec2 mouse sera that showed a predominant homogenous cytoplasmic staining pattern was ~65%, while the remaining 35% showed a predominant homogenous nuclear staining pattern characteristic of, but not confirmed for, anti-SS-A/Ro and anti-SS-B/La autoantibodies. In contrast, only ~40% of the sera from C57BL/6.NOD-Aec1Aec2.C3–/– mice showed a presence of autoantibodies, 30% of which were anticytoplasmic and only 10% antinuclear. Interestingly, the staining patterns of autoantibodies in the sera of C57BL/6.NOD-Aec1Aec2.C3–/– mice exhibited a shift in the Ab-staining patterns that matched those seen in the nondiseased C57BL/6 mice. Clearly, identifying which ANAs are present in the C57BL/6.NOD-Aec1Aec2 mice will require further in-depth analyses.


Figure 5
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  		FIGURE 5. Identification of the autoantibodies patterns in sera of C57BL/6, C57BL/6.NOD-Aec1Aec2, and C57BL/6.NOD-Aec1Aec2.C3–/– mice. A, Representative patterns of cellular staining of Hep2 cells by sera prepared from C57BL/6.NOD-Aec1Aec2 (n = 33), C57BL/6.NOD-Aec1Aec2.C3–/– (n = 24), and C57BL/6 (n = 11) mice. Fixed HEp-2 substrate slides were incubated with individual mouse sera diluted 1/40, followed by development with FITC-conjugated goat anti-mouse IgG. Fluorescent patterns were detected by fluorescence microscopy at x200 magnification. B, Percentage of individual mouse sera exhibiting the specific patterns of staining. Statistical analysis was performed using {chi}2 test. ***, p < 0.001.

 
Another second set of important autoantibodies, whose appearance is believed to be directly involved in the shutdown of salivary and lacrimal gland secretion, is the anti-muscarinic acetylcholine receptor Abs, in particular those reactive against the M3R (26). Previous studies with NOD.IL4–/– and NOD.B10-H2b.IL4–/– mice indicated that IgG1 isotype-specific anti-M3R autoantibodies were the more important autoantibody, acting as potential effectors of secretory dysfunction observed in submandibular and lacrimal glands (19, 22). To determine the presence of anti-M3R Abs, aliquots of sera pooled from appropriate groups were incubated with mouse M3R-transfected Flp-In CHO cells, tagged using FITC-conjugated anti-isotype-specific secondary Abs, and subsequently analyzed by flow cytometry (26). As shown in Fig. 6, sera from C57BL/6.NOD-Aec1Aec2 mice proved positive for IgG1, IgG2b, and IgG2c anti-M3R autoantibodies. In contrast, sera from both C57BL/6 and C57BL/6.NOD-Aec1Aec2.C3–/– mice showed a loss or decline in IgG1, IgG2b, and IgG2c anti-M3R levels as indicated by decreased mean fluorescent intensities. Although changes in mean fluorescent intensities for the IgG Abs are relatively small and do not usually reach statistical significance, it is important to recognize that the titers of IgG anti-M3R autoantibodies are known to be low, even in the NOD mouse considered to produce the highest titers.


Figure 6
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  		FIGURE 6. Detection of anti-M3R Abs. Pooled sera collected from C57BL/6 (27 wk of age), C57BL/6.NOD-Aec1Aec2 (30 wk of age), and C57BL/6.NOD-Aec1Aec2.C3–/– (24 wk of age) mice. Mouse M3R-transfected Flp-In CHO cells were incubated with either a pooled sera or respective isotype control sera. Cells were washed with FACS buffer, resuspended in 50 µl of FACS buffer and developed with either FITC-conjugated goat anti-mouse IgM, IgG1, IgG2b, IgG2c, or IgG3, then analyzed using a FACScan cytometer. M (marker) values equal mean fluorescent indices.

 
Similar to observations in humans where nearly all sera contain IgM anti-M3R autoantibodies (T. Cornelius, R. Jonsson, and A. B. Peck, our unpublished observation), all three mouse strains produced IgM anti-M3R autoantibody. The presence of IgM anti-M3R Abs in both healthy individuals and SjS patients may indicate a protective rather than pathogenic mechanism. In contrast, all sera were negative for IgA, IgD, and IgE anti-M3R Abs (data not shown).

Lack of salivary gland dysfunction in C57BL/6-NOD.Aec1Aec2.C3–/– mice

With onset of SjS-like disease in C57BL/6-NOD.Aec1Aec2 mice, a number of changes in glandular physiology and function become apparent. These include loss of saliva and/or tear secretion, marked changes in protein content of saliva and tear secretions, loss of major enzyme activities (e.g., {alpha}-amylase), and reduced levels of important growth and antimicrobial factors (e.g., epidermal growth factor-beta and PSP, respectively). Such temporal changes correlate with the appearance of leukocyte infiltrates within the exocrine glands, loss of acinar cell mass, and hyperplasia of ductal cells (27, 28). To measure temporal changes in stimulated saliva flow rates, individual male and female C57BL/6-NOD.Aec1Aec2, C57BL/6-NOD.Aec1Aec2.C3–/–, and C57BL/6 mice at appropriate ages were injected with isopreterenol and pilocarpine, their saliva secretions collected, and the collected saliva volumes standardized to the weight of the animal. As shown in Fig. 7, both female and male C57BL/6-NOD.Aec1Aec2 mice displayed a chronic loss of stimulated saliva secretions (46%; p < 0.001 and 36%; p < 0.001, respectively) between the ages of 4 and 25 wk. Interestingly, male and female C57BL/6.NOD-Aec1Aec2.C3–/– mice, like C57BL/6 mice, exhibited no statistically significant loss of stimulated saliva excretions over the same time period.


Figure 7
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  		FIGURE 7. Temporal changes in stimulated saliva flow rates of C57BL/6, C57BL/6.NOD-Aec1Aec2, and C57BL/6.NOD-Aec1Aec2.C3–/– female (A) and male (B) mice. Stimulated saliva flow rates are presented for sets of mice selected within each experimental group following injections of isopreterenol/pilocarpine solution at the times indicated in the figures. Saliva was collected for 10 min from the oral cavity using a micropipette starting 1 min after injection of the secretagogue. The volumes of individual collections were determined and the mean of collections within each group determined. **, p < 0.01; ***, p < 0.001.

 
Two important indicators of salivary gland dysfunction are the gain-loss protein concentration and the reduced {alpha}-amylase activity in secreted saliva (30). As presented in Table I, comparison of protein concentrations in prediseased (8 wk of age) vs diseased (>24 wk of age) C57BL/6.NOD-Aec1Aec2 mice revealed a slight increase in overall protein concentration. Previous studies (30) have shown that the levels of both {alpha}-amylase activity and PSP, two of the more abundant proteins in saliva, are greatly reduced over this time frame. In contrast, the saliva collected from both C57BL/6 and C57BL/6.NOD-Aec1Aec2.C3–/– mice actually showed modest temporal increases in protein concentrations.

To determine whether {alpha}-amylase activity is affected in the C3 gene KO mice, saliva samples from C57BL/6, C57BL/6.NOD-Aec1Aec2, and C57BL/6.NOD-Aec1Aec2.C3–/– mice were analyzed using a commercially available kit in which {alpha}-amylase activity is measured by degradation of ethylidene-pNP-G7. As presented in Table I, saliva from diseased C57BL/6.NOD-Aec1Aec2 mice exhibited reduced enzyme activity when compared with their saliva collected during the prediseased stage. Saliva collected from C57BL/6 mice, as well as C57BL/6.NOD-Aec1Aec2.C3–/– mice, as expected, exhibited either no change in or slightly increased levels of {alpha}-amylase activity over this same time frame. These data indicate that the end-stage pathophysiological changes are dependent on both the activation of the autoimmune response and the aberrant physiological and biochemical preimmune activities that apparently initiate an immune response.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we have examined the role of C3 in the development and onset of SjS-like disease by genetically disrupting the C3 gene in the C57BL/6.NOD-Aec1Aec2 mouse, a congenic strain of the NOD model of autoimmune exocrinopathy. Results presented here clearly reveal that C3 has a major and widespread impact on both the development and onset phases of SjS-like disease associated with NOD mice. By eliminating the C3 gene in the C57BL/6.NOD-Aec1Aec2 mouse, many preclinical markers and most clinical manifestations of SjS-like disease were altered or totally absent in C57BL/6-NOD.Aec1Aec2.C3–/– mice. These included reductions in acinar cell apoptosis, concomitantly reduced levels of cleaved products of caspase-3, maintenance of normal glandular architecture, lack of leukocyte infiltration into the submandibular glands, reductions in the synthesis of autoantibodies, and most importantly, retention of normal saliva secretion in both female and male animals. In addition, C57BL/6-NOD.Aec1Aec2.C3–/– mice did not exhibit increased numbers of MZ B cell subpopulations, compared with C57BL/6-NOD.Aec1Aec2 mice, yet at the same time retained some early pathological manifestations, as shown by the activation of serine kinases with proteolytic activity on the major antibacterial protein, PSP, and slightly elevated levels of MMP-9 possibly indicating active ECM remodeling.

Although a direct role for complement in disease progression has been established for a number of autoimmune diseases, in particular systemic lupus erythematosus and rheumatoid arthritis, a similar role in SjS remains controversial despite the general concept that complement plays little or no role. Early studies (33) suggested a correlation between deposition of IgA and/or IgM immune complexes and clinical manifestations of SjS; however, Cuida et al. (37) reported the presence in both saliva and salivary glands of SjS patients of complement regulatory proteins, including protectin (CD59), decay accelerating factor (CD55), membrane cofactor protein (CD46), and clusterin (SP-40; Ref. 38), that prevented complement activation on the tissues. Recent reports now indicate that hypocomplementemia, specifically reduced levels of C3 and/or C4, is associated with B cell lymphoma development and increased pathogenicity in SjS, thereby serving as one of the strongest predictors for unfavorable long-term outcome (39). Unfortunately, these studies are restricted to SjS patients examined at the clinical phase of the disease, considered to be equivalent to phase III disease in our animal models of SjS, a time at which complement activation may no longer play a relevant role in the development of SjS-like disease. Considering the heterogeneity of any SjS patient population, it is not surprising that there is considerable inconsistency in the data collected and interpreted. Thus, despite the fact that the SjS-like disease profiles in our inbred mouse models are also far from homogeneous, our current study, together with our earlier study in which injections of CVF was found to suppress onset of clinical disease (24), clearly points to the importance of complement in the earlier stages of disease development.

To date, we have found no evidence to suggest that the effects of complement in SjS-like disease are due to activation of the membrane attack complex of complement. First, parental strain NOD mice are deficient in C5, yet clearly exhibit a strong SjS disease phenotype (40). Furthermore, immunostaining of histological sections of exocrine glands in the NOD and NOD-derived mice have thus far shown no detectable depositions of C3 (our unpublished observations), suggesting that complement-mediated lysis of acinar tissues is not a major mechanism of glandular dysfunction. In the present study, we have used the C57BL/6-NOD.Aec1Aec2 mouse model, a mouse strain whose background genetics is C57BL/6J and thus not complement deficient; yet, despite this, there are no indications that the disease phenotype of C57BL/6-NOD.Aec1Aec2 mice is different than that of NOD mice. Thus, the decreased levels of preclinical pathology and subsequent clinical disease in C57BL/6-NOD.Aec1Aec2.C3–/– mice point to multiple functional roles for complement. This is an unexpected result as we had hypothesized originally that C3 would be involved primarily in the activation of the autoreactive B cell populations via the C3d component.

In addition to its role in the activation of B lymphocytes, C3 is important in enhancing inflammatory responses, recruitment of phagocytes to a site of injury, opsonization of microorganisms, and removal of immune complexes (38). Because SjS is a disease in which B lymphocyte is critical for a number of preclinical and clinical manifestations, including a state of B cell hyperproliferation with sharp increases in autoantibody production (41), we have speculated that C3, in particular C3d, is capable of lowering the threshold for the activation of autoreactive B cells due to cross-linking of B cell receptors and their coreceptors, CD19 and CD21, thereby modulating the strength, intensity, and duration of the signal generated by BCRs (42). Signals generated by the coligation of CD19/CD21 and BCR through the C3d fragment bridging act as positive regulators of B cell activation, contributing directly to the hyperproliferative and hyperactive properties of autoreactive B cells in SjS (13). Therefore, elimination of C3/C3d could prevent formation of functional germinal centers in secondary lymphoid tissues, as well as formation of ectopic germinal center-like foci, often found in the exocrine glands of human SjS patients and our animal models of SjS-like disease (24).

Beyond this interaction of C3/C3d with BCRs and coreceptors, complement directly influences innate immunity. Innate immunity is responsible, in part, for the evolution of specific pattern recognition proteins that activate complement by binding C3d, which then binds to or forms complexes with self or non-self Ags to facilitate and enhance inflammatory and immunological responses via interaction with specific complement receptors, such as CR1/CR2 present on follicular dendritic cells (43). Such localization of Ags by C3d and complement receptors on follicular dendritic cells in secondary lymphoid tissues promotes Ag uptake and presentation. As a result, activation of FDC by the innate immune system impacts the adaptive immune response by enhancing formation of germinal centers, recruiting and retaining B lymphocytes within germinal centers for optimal survival and activation, as well as subsequent Ab production (44). Interestingly, our current findings suggest that in order for the full initiation of an autoimmune response leading to SjS, the complement system is required during the innate response phase that establishes an antigenic and immunological basis for the adaptive immune response to produce pathogenic Abs. As the result, elimination of C3, while not affecting the intrinsic genetic predisposition to autoimmunity (e.g., activation of serine kinases and MMP involved in tissue remodeling), significantly reduces the overall immune response and tissue destruction (e.g., leukocyte migrations and production of autoantibodies).

Lastly, C3 appears to play an important role in the establishment of cellular compositions within the secondary lymphoid organs, in particular the spleen, by impacting the selection and maturation of B cell subpopulations. Weak BCR signaling tends to induce the maturation of MZ B cells, while strong BCR signaling favors maturation of FO B cells (45, 46). In the present study, C57BL/6.NOD-Aec1Aec2.C3–/– exhibited a decreased number of MZ B cells in both male and female mice when compared with age-and sex-matched disease-prone C57BL/6.NOD-Aec1Aec2 mice. This finding raises the possibility that without C3, autoantigens in low abundance are no longer capable of stimulating autoreactive B cells thought to reside in the MZ (47). Current studies are attempting to address this issue.

In summary, results obtained in this study clearly reinforce the importance of C3 in the pathogenesis of SjS-like disease in the C57BL/6.NOD-Aec1Aec2 mouse model. Whether C3 can be shown to play an equally important role in human SjS disease may only be possible when individuals predisposed to developing SjS can be identified before onset of clinical disease. Nevertheless, the use of animal models like C57BL/6.NOD-Aec1Aec2 and C57BL/6.NOD-Aec1Aec2.C3–/– to identify mechanisms by which complement helps initiate autoimmunity, e.g., by establishing an early innate inflammatory response or binding self-Ag to lower the threshold for BCR signaling, should provide new insights into the human disease and experimental designs for future studies. Recent determinations of the crystal structure for activated complement protein C3b (48, 49, 50) reinforce the importance of C3 and its cleaved products in autoimmune diseases, thus expanding the possible development of therapeutic strategies for intervening in human autoimmune diseases during their preclinical phases.


   Acknowledgments
 
We thank Marievic Bulosan for her technical help during these studies. We also thank Dr. Edward Chan for helpful discussion concerning ANA staining.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported in part by Public Health Service (PHS) Grants DE013769, DE014344, and DE015152 (to A.B.P.) from the National Institutes of Health. C.Q.N. was supported by a postdoctoral fellowship from PHS Grant T32 DE07200. Back

2 Address correspondence and reprint requests to Dr. Cuong Q. Nguyen, Department of Oral Biology, College of Dentistry, University of Florida, P.O. Box 100424, Gainesville, FL 32610. E-mail address: Nguyen{at}pathology.ufl.edu Back

3 Abbreviations used in this paper: SjS, Sjögren’s syndrome; CVF, cobra venom factor; C3, complement component-3; KO, knockout; PSP, parotid secretory protein; DAPI, 4',6'-diamidino-2-phenylindole; ANA, anti-nuclear autoantibody; MMP, matrix metalloproteinase; M3R, muscarinic acetylcholine type 3 receptor; ECM, extracellular matrix; FO, follicular; MZ, marginal zone. Back

Received for publication March 8, 2007. Accepted for publication June 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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