Passive transfer of antibodies to the linear epitope 60 kD Ro 273-289 induces features of Sjögren's syndrome in naive mice

Abstract

Sjögren's syndrome (SS) is an autoimmune inflammatory disease that primarily affects the lacrimal and salivary glands causing dry eyes and mouth. Antibodies to Ro60 are observed frequently in patients with SS; however, the role of these antibodies in SS initiation and progression remains unclear. The sequence Ro60 273-289 (Ro274) is a known B cell epitope of Ro60 and antibodies to this epitope have been observed in a subset of SS patients and in animals immunized with Ro60 protein. Animals immunized with Ro274 linear peptide develop a Sjögren's-like illness. We hypothesized that passive transfer of anti-Ro274-specific immunoglobulin (Ig)G would induce a Sjögren's-like phenotype. To evaluate this hypothesis, we adoptively transferred affinity-purified Ro274 antibodies into naive BALB/c animals, then evaluated salivary gland histology, function and IgG localization 4 days post-transfer. At this time-point, there was no demonstrable mononuclear cell infiltration and salivary glands were histologically normal, but we observed a functional deficit in stimulated salivary flow of animals receiving Ro274 antibodies compared to animals receiving control IgG. Cellular fractionation and enzyme-linked immunosorbent assay revealed Ro274-specific antibodies in the nucleus and cytoplasmic fractions of isolated parotid salivary gland cells that was confirmed by immunohistochemistry. These data support the hypothesis that antibodies to Ro274 deposit in salivary glands can enter intact salivary gland cells and are involved in the dysregulation of salivary flow in SS.

Keywords: antigens/peptides/epitopes; autoantibodies; autoimmunity; rodent; systemic lupus erythematosus.

Figure 1

Immunization with 60 kD Ro274–290 peptide promotes formation of anti-Ro274 antibodies in BALB/c mice. (a) Sera from individual BALB/c mice (n = 5/group) immunized with Ro273–289 peptide (▪) or saline (▴) were tested in duplicate by enzyme-linked immunosorbent assay (ELISA) for antibodies to a Ro273–289 MAP peptide, as described previously. Individual points on the graph represent data obtained from a single mouse. (b) The mean and standard deviation of anti-Ro274 antibody in sera of Ro274-immunized (black bar) compared to saline-immunized (grey bar) mice (P < 0·0001; Student's t-test).

Figure 2

Immunoglobulin (IgG) antibodies are detected in salivary glands of naive BALB/c mice following Ro274 IgG adoptive transfer. (a) Haematoxylin and eosin (H&E) paraffin sections of salivary tissue from mice 4 days after receiving adoptively transferred anti-Ro274 antibodies at ×20 and ×40 total magnification (top left and bottom left, respectively), and mice receiving control IgG at ×20 and ×40 total magnification (top right and bottom right, respectively). White bar represents 100 μm. (b) Ig deposition in salivary gland cryosections 4 days post-anti-Ro274 (left) and control (right) IgG transfer to naive BALB/c mice assessed by immunofluorescence staining at ×40 total magnification. White bar represents 100 μm.

Figure 3

Anti-Ro274 antibodies are detectable in the serum and in the nucleus and cytoplasm of salivary gland acinar cells by enzyme-linked immunosorbent assay (ELISA). (a) Anti-Ro274 titres were determined for cytoplasmic and nuclear extracts of salivary gland acinar cells and serum by ELISA on days 1, 17, 38, 60 and 78 in mice immunized with Ro274–290 peptide. (b) Anti-Ro274 titres in cytoplasmic and nuclear extracts of salivary gland acinar cells and serum were determined for mice immunized with saline at days 17 and 86. (c) The nuclear : cytoplasmic anti-274 antibody ratio was assessed for both Ro274 peptide and saline-immunized mice.

Figure 4

Immunoglobulin (IgG) antibodies are visible in the nucleus, cytoplasm and interstitium in salivary gland tissue of anti-Ro274 recipients. IgG antibodies were detected using donkey anti-mouse IgG-Alexa 647 (red). Cell membranes were delineated using wheat germ agglutinin (green) and cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (a) Negative section of salivary gland (×400 total magnification). White bar represents 20 μm. (b) Intranuclear IgG staining (white arrow) observed in acinar cells (×630 total magnification). White bar represents 10 μm. (c,d) Interstitial and cytoplasmic IgG staining can be seen throughout the salivary gland tissue (×400 total magnification). White bar represents 20 μm.

Figure 5

Median salivary flow is decreased in naive BALB/c mice receiving adoptively transferred anti-Ro274 antibodies. Median, minimum, maximum, 75th and 25th percentile salivary flow was assessed in naive BALB/c mice 4 days following adoptive transfer of control (left) immunoglobulin (IgG) antibodies or anti-Ro274 (right) antibodies (n = 5/group). Salivary flow is decreased significantly in naive BALB/c mice receiving anti-Ro274 antibodies compared to those receiving control IgG (*P = 0·0317, Mann–Whitney U-test).