Dana Cavill

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

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 Sjögrens 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.

Antibodies Raised Against the Second Extracellular Loop of the Human Muscarinic M3 Receptor Mimic Functional Autoantibodies in Sjögren's Syndrome

Functional antimuscarinic M3 receptor (M3R) autoantibodies have been shown to inhibit cholinergic neurotransmission at the postsynaptic level and appear to mediate parasympathetic dysfunction, including sicca symptoms in Sjögrens syndrome (SS). The precise epitope(s) involved in the inhibition of M3R‐mediated cholinergic neurotransmission has not been defined. In this study, an active immunization approach to raise antibodies with functional activity against the second extracellular loop of the M3R was used and their functional properties were compared with those of human autoantibodies. Peptides corresponding to the second extracellular loop of the M3R were used as immunogens in rabbits, and antisera were tested for inhibition of carbachol‐evoked colon smooth muscle contraction in parallel with immunoglobulin G from a patient with SS. Anti‐M3R antibodies were affinity purified on a peptide representing a dominant functional epitope at the COOH terminus of the second extracellular loop of the M3R and tested for concentration‐dependent inhibition. Experimentally raised anti‐M3R antibodies, like the human autoantibodies, showed concentration‐dependent and noncompetitive inhibition of carbachol‐evoked colon contractions. Inhibitory activity was detected by functional assays at concentrations as low as 3 ng/ml, which was below the threshold of detection of antibody by peptide enzyme‐linked immunosorbent assay. It is concluded that the experimentally raised anti‐M3R antibodies share the functional properties of autoantibodies in patients with SS.

Tolerance through Indifference: Autoreactive B Cells to the Nuclear Antigen La Show No Evidence of Tolerance in a Transgenic Model

Systemic autoimmune diseases are characterized by the production of high titer autoantibodies specific for ubiquitous nuclear self-Ags such as DNA, Sm, and La (SS-B), so the normal mechanisms of B cell tolerance to disease-associated nuclear Ags have been of great interest. Mechanisms of B cell tolerance include deletion, anergy, developmental arrest, receptor editing, and B cell differentiation to the B-1 subtype. However, recent studies in our laboratory have suggested that B cell tolerance to the nuclear autoantigen La is limited in normal mice, and tolerance may reside primarily in the T cell compartment. To test this hypothesis, we created Ig transgenic mice expressing the IgM H chain from an mAb specific for a xenogeneic epitope within human La (hLa). These mice were bred with hLa-transgenic mice that constitutively express hLa in a manner comparable to endogenous mouse La. Between 5–15% of transgenic B cells developing in the absence of hLa were specific for hLa, and these cells were neither depleted nor developmentally arrested in the presence of endogenous hLa expression. Instead, these autoreactive B cells matured normally and differentiated into Ab-forming cells, capable of secreting high titer autoantibody. Additionally, the life span of autoreactive hLa-specific B cells was not reduced, and they were phenotypically and functionally indistinguishable from naive nonautoreactive hLa-specific B cells developing in the absence of hLa. Together these data suggest a lack of intrinsic B cell tolerance involving any known mechanisms indicating that these autoreactive B cells are indifferent to their autoantigen.

Vasoactive intestinal peptide binding autoantibodies in autoimmune humans and mice

Autoantibodies capable of binding the immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) were detected in the sera of a mouse strain prone to autoimmune disease due to the lpr mutation (MRL/lpr). The autoantibodies were not present in control wildtype MRL/lpr mice, but they were readily detected in humans without autoimmune disease. The binding was due to low affinity VIP recognition. Increased VIP binding activity was evident in patients with systemic lupus erythematosus but not systemic sclerosis, Sjögrens syndrome (SS), rheumatoid arthritis or autoimmune thyroiditis. Recombinant VIP binding Fv clones (fragment variable; the variable domains of the light and heavy chains antibody subunits joined with a peptide linker) were isolated from a phage display library prepared from lupus patients. One Fv clone displaying VIP-selective binding and several clones displaying cross-reactivity with unrelated peptides were identified. Replacement mutations in the VIP-selective clone were preferentially localized in the regions known to make contacts with the antigen, i.e. the complementarity determining regions, suggesting that the selective binding activity is due to immunological maturation of the antibodies. Frequent occurrences of autoantibody responses to VIP indicate that immunological tolerance to this neuropeptide can be readily broken. The depletion of VIP by specific antibodies in autoimmune disease may interfere with VIP regulation of T cells and inflammatory cells and result in further amplification of autoreactive immunological responses.

Restricted Specificity of Intermolecular Spreading to Endogenous La (SS‐B) and 60 kDa Ro (SS‐A) in Experimental Autoimmunity

Intermolecular spreading of humoral autoimmunity to different components of the Ro (SS‐A) and La (SS‐B) ribonucleoprotein (RNP) complex has been reported following immunization with a single component of the complex. Although the immune response to the immunizing antigen is polyclonal and diversified, little is known about the specificity of the recruited autoimmune responses to the endogenous Ro and La antigens which drive B‐cell spreading. To determine the specificity of intermolecular spreading to La, we examined sera from 52 kDa Ro (Ro52)‐ and 60 kDa Ro (Ro60)‐immunized C3H/HeJ mice for reactivity with recombinant fragments spanning endogenous mouse (m)La by enzyme‐linked immunosorbent assay (ELISA) and immunoblotting. Sera from mice primed and boosted with recombinant Ro52 and Ro60 showed reactivity restricted to the COOH‐terminal fragment of mLa (aa361–415). The recruited anti‐La response was species‐specific, cross‐reacting weakly with the corresponding region on the human La molecule, and was abrogated by the preabsorption of the Ro‐immune sera with mLa 361–415. Analogous experiments using recombinant mRo60 fragments spanning the mRo60 molecule revealed a similar pattern of oligoclonality in the specificity of anti‐Ro60 autoimmunity following active immunization with La and Ro52. These results suggest that intermolecular–intrastructural T–B help is limiting in this model, and reveal unsuspected immunodominance of selected Ro–La epitopes in the spreading of the autoantibody response to these structures. The focusing of the recruited autoantibody response to these COOH‐terminal regions of the Ro and La polypeptides may also reflect the surface accessibility of these regions in La–Ro RNP.