Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren's syndrome

Abstract

BAFF (BLyS, TALL-1, THANK, zTNF4) is a member of the TNF superfamily that specifically regulates B lymphocyte proliferation and survival. Mice transgenic (Tg) for BAFF develop an autoimmune condition similar to systemic lupus erythematosus. We now demonstrate that BAFF Tg mice, as they age, develop a secondary pathology reminiscent of Sjögren's syndrome (SS), which is manifested by severe sialadenitis, decreased saliva production, and destruction of submaxillary glands. In humans, SS also correlates with elevated levels of circulating BAFF, as well as a dramatic upregulation of BAFF expression in inflamed salivary glands. A likely explanation for disease in BAFF Tg mice is excessive survival signals to autoreactive B cells, possibly as they pass through a critical tolerance checkpoint while maturing in the spleen. The marginal zone (MZ) B cell compartment, one of the enlarged B cell subsets in the spleen of BAFF Tg mice, is a potential reservoir of autoreactive B cells. Interestingly, B cells with an MZ-like phenotype infiltrate the salivary glands of BAFF Tg mice, suggesting that cells of this compartment potentially participate in tissue damage in SS and possibly other autoimmune diseases. We conclude that altered B cell differentiation and tolerance induced by excess BAFF may be central to SS pathogenesis.

Figure 1

Enlarged and inflamed salivary glands in BAFF Tg mice. (a) Mice 15–17 months old (four control littermates and four BAFF Tg mice) were sacrificed the same day for organ collection. Both right and left submaxillary glands were collected and weighed. The data shows the combined weight (mean ± SD) of both glands. These results are representative of at least four separate groups of dissected age-matched animals. (b) Paraffin sections of submaxillary glands from a control littermate (left panel) and two BAFF Tg mice (middle and right panels) were stained with hematoxylin and eosin. Arrows indicate ducts and acinar cells in the left and right panels. The arrow in the middle panel shows acinar destruction. Asterisks indicate periductal infiltrates (foci). Magnification: ×100. (c) Paraffin sections of submaxillary glands from seven control mice and 22 BAFF Tg mice (12–17 months old) were prepared as shown in b and scored for disease as described in Methods. Bars indicate the mean disease score for each group. *P < 0.05, **P < 0.03.

Figure 2

Identification of MZ-like B cells infiltrating salivary glands of BAFF Tg mice. Lymphocytes were isolated from salivary glands of BAFF Tg mice (13–17 months old) and control littermates. Cells were stained with anti-B220, anti-CD5, anti-IgM, and anti-CD43 using multi-color flow cytometry. (a) Gates used to identify B220^hi and B220^lo/int B cells in control and BAFF Tg mice. (b) Expression of IgM and CD5 on B220^hi cells and B220^lo/int cells from BAFF Tg and control mice. The IgM^hi B cell gate on B220^hi cells is boxed and the B-1a and B-1b gates on B220^lo/int cells are indicated. (c) Expression of CD43 on gated B220^hi/IgM^hi cells and gated B-1a and B-1b cells from a BAFF Tg mouse. (d) L-selectin expression on lymphocytes from salivary gland and inguinal lymph node of a BAFF Tg mouse, gated on B220^hi/IgM^hi cells and on IgM⁺ cells. (e) Lymphocytes from a BAFF Tg mouse and a control littermate were prepared as in a and stained with anti-B220, anti-IgM, and antibodies to the indicated markers. Cells were gated on B220^hi and IgM^hi cells, as shown in the top histograms. In c–e, mean fluorescence intensity (MFI) and bars delineating negative control staining are indicated. (f) Schematic representation of the common and distinct markers expressed on MZ B cells (right circle), B-1 cells (left circle), and T1 B cells (lower circle). The phenotype of the B cells infiltrating submaxillary glands of BAFF Tg mice is represented by the dotted ellipse. These results are representative of 12 BAFF Tg mice and seven control mice analyzed. FSC, forward light scatter.

Figure 3

Decreased saliva flow in older BAFF Tg mice. Thirteen BAFF Tg mice (diamonds) and 14 control littermates (circles) were injected with pilocarpine prior to saliva collection as described in Methods. (a) Mice 13–15.5 months old. (b) Mice 8–10 months old. Mean values for saliva flow are shown with a bar. P values are indicated in each panel.

Figure 4

Elevated levels of BAFF in sera and salivary gland tissues from patients suffering from primary SS; no correlation with levels of total IgG, RF, and presence of anti-Ro/La autoantibodies. (a) Individual serum BAFF levels in 39 healthy controls (squares), 41 patients with primary SS (diamonds), 53 patients with SLE (circles), and 53 patients with RA (triangles) were measured by ELISA. The horizontal black bars indicate the mean for each group: normal 10.4 ± 13 (ng/ml); SS 53 ± 67 (ng/ml); SLE 12.7 ± 24.4 (ng/ml); and RA 23 ± 47 (ng/ml). Sera of 16 normal individuals, 6 SS, 20 SLE, and 10 RA patients had no detectable levels of BAFF. The dotted line delineates the range of normal BAFF levels. *P < 0.04, as determined by ANOVA. (b and c) Correlation of serum BAFF levels in patients with SS with the levels of IgG (b) and RF (c) in each serum sample. R and P values were calculated by ANOVA. (d) Levels of BAFF in patients with anti-Ro plus anti-La or anti-Ro only, and in patients with no precipitin detected (Neg). (e) Paraffin sections of a human labial gland biopsy from a patient with SS were stained with anti-human BAFF antibody or an isotype-matched control antibody. Magnification: ×200. Staining of normal human labial gland with anti-human BAFF antibody is also shown (magnification: ×100). These pictures are representative of four patients with primary SS and three control tissues analyzed. RF, rheumatoid factors.