Development of Sjogren's syndrome in nonobese diabetic-derived autoimmune-prone C57BL/6.NOD-Aec1Aec2 mice is dependent on complement component-3

Abstract

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.

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 EAVPQNLN LDVELLQQ 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.

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.

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).

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 (CD21^highCD23^int), and FO (CD21^intCD23^low) 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.

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 ×200 magnification. B, Percentage of individual mouse sera exhibiting the specific patterns of staining. Statistical analysis was performed using χ² test. ***, p < 0.001.

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.

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.