Neutralization of muscarinic receptor autoantibodies by intravenous immunoglobulin in Sjögren syndrome.

Autoantibodies that inhibit M3 muscarinic receptor (M3R)-mediated neurotransmission and cause bladder and bowel dysfunction have been reported in patients with Sjögren syndrome and belong to a family of functional autoantibodies that includes the thyroid-stimulating hormone receptor antibody present in Graves disease. We have recently reported that antiidiotypic antibodies present in pooled immunoglobulin (Ig) G or IgG from healthy individuals neutralize anti-M3R antibody-mediated inhibition of smooth muscle contraction in vitro. Here we extend these studies to the clinic by examining whether therapeutic doses of intravenous immunoglobulin (IVIG) provided to patients with autoimmune diseases neutralize anti-M3R activity in vivo and improve bladder and bowel symptoms. Three patients with primary Sjögren syndrome, dermatomyositis, and celiac disease, respectively, all of whom had anti-M3R activity on a functional bladder contractile assay, were provided a single course of IVIG at a dose of 400 mg/kg per day for 5 days. Anti-M3R activity was neutralized at 4 weeks after IVIG infusion, whereas levels of specific autoantibodies (anti-La, anti-Jo-1, and anti-tissue transglutaminase) were unchanged. Bladder and bowel scores revealed variable improvement after IVIG. Neutralization of anti-M3R activity by IVIG in vivo, presumably as a result of antiidiotypic antibodies directed specifically against anti-M3R autoantibodies, provides a clinical correlate of our in vitro findings. This offers a rationale for IVIG as a treatment for parasympathetic dysfunction in patients with autoantibodies inhibiting postganglionic cholinergic neurotransmission. We suggest the presence of a network of naturally occurring antiidiotypic antibodies that regulate the expression of functional autoantibodies against neuronal receptors and ion channels.

Passive transfer of Sjogren's syndrome IgG produces the pathophysiology of overactive bladder.

OBJECTIVE: The presence, in patients with primary and secondary Sjogren's syndrome (SS), of autoantibodies that acutely inhibit M(3) muscarinic receptor (M3R)-mediated bladder contractions is difficult to reconcile with the fact that symptoms of detrusor overactivity and other features of cholinergic hyperresponsiveness occur in this disease. This study was undertaken to examine the in vivo effects of these autoantibodies on bladder function by examining bladder responsiveness and compliance following passive transfer of patient IgG to mice. METHODS: Contractile responses of isolated bladder strips both to the muscarinic agonist carbachol and to electrically evoked acetylcholine release were measured 48 hours after injection of mice with patient or control IgG. A whole bladder assay with intact neuronal pathways was developed to assess bladder wall compliance on filling cystometry. Expression of M3R in bladders from IgG-injected mice was assessed by immunohistochemistry. RESULTS: Passive transfer of SS IgG with inhibitory anti-M3R activity produced a paradoxical increase in contractile responses of detrusor strips to cholinergic stimulation. Cystometry of whole bladders revealed a corresponding decrease in bladder wall compliance and phasic detrusor contractions upon filling, replicating the urodynamic features of an overactive bladder. The features of cholinergic hyperresponsiveness were associated with increased postsynaptic M3R expression and were reproduced by injecting mice with a rabbit antibody against the second extracellular loop of M3R. CONCLUSION: These findings are consistent with the notion that there is initial inhibition of parasympathetic neurotransmission by antagonistic autoantibodies to M3R, which produces a compensatory increase in M3R expression in vivo. The enhanced cholinergic responses during bladder distention result in detrusor overactivity. We conclude that the overactive bladder associated with SS is an autoantibody-mediated disorder of the autonomic nervous system, which may be part of a wider spectrum of cholinergic hyperresponsiveness.

Antiidiotypic antibodies neutralize autoantibodies that inhibit cholinergic neurotransmission.

OBJECTIVE: Functional autoantibodies that inhibit M(3) muscarinic receptor (M3R)-mediated neurotransmission have been reported in patients with Sjögren's syndrome (SS) and in patients with scleroderma. Because of limited reports that intravenous immunoglobulin (IVIG) improves dysautonomia in primary SS, we investigated whether IVIG neutralizes the effect of anti-M3R antibodies on colon smooth muscle contractions, in an in vitro functional assay. METHODS: IgG obtained from patients with primary SS, patients with rheumatoid arthritis and secondary SS, and patients with scleroderma was tested, before and after coincubation with equimolar amounts of IVIG or its F(ab')(2) and Fc fractions, for the ability to inhibit carbachol-evoked colon smooth muscle contractions. In addition, patient IgG was passed through an IVIG F(ab')(2) column, and unretained IgG was tested for functional activity on colon smooth muscle strips. Purified IgG obtained from healthy adults was also examined for a neutralizing effect on anti-M3R antibody activity. RESULTS: Inhibition of colon contractions was mediated by the Fab fraction of patient IgG. Coincubation of IgG from the 3 patient groups with IVIG or its F(ab')(2) fragment neutralized anti-M3R antibody-mediated inhibition of cholinergic smooth muscle contractions. Preabsorption of patient IgG with Sepharose-bound IVIG F(ab')(2) removed the anti-M3R inhibitory activity. In addition, purified IgG from each of 4 healthy adults neutralized the functional autoantibodies. CONCLUSION: Anti-M3R antibody activity does not require receptor crosslinking. Antiidiotypic antibodies present in pooled IgG neutralize patient IgG-mediated inhibition of M3R cholinergic neurotransmission, providing a rationale for IVIG as a treatment of autonomic dysfunction in patients with SS and patients with scleroderma. Furthermore, antiidiotypic antibodies in healthy individuals may prevent the emergence of pathogenic anti-M3R autoantibodies.

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.

Anti-La/SSB antibodies transported across the placenta bind apoptotic cells in fetal organs targeted in neonatal lupus.

OBJECTIVE: To determine whether La and/or Ro epitopes on apoptotic cells in fetal organs that are targeted in neonatal lupus syndrome (NLS) are accessible for binding by autoantibodies in vivo, we traced the fate of transplacental autoantibodies in a murine passive transfer model. METHODS: Pregnant mice at day 15 of gestation (E15) were injected intraperitoneally with human anti-Ro/La-positive sera or control sera, and transplacental transfer of human autoantibodies was tested by enzyme-linked immunosorbent assay with recombinant antigens. Multiple cryostat sections at the level of the heart of E17 fetuses were visualized simultaneously for human IgG binding and apoptosis (TUNEL) under confocal microscopy. Serial paraffin sections of E17 and E19 fetuses were examined for histologic evidence of inflammation. RESULTS: Human IgG anti-52-kd Ro, anti-60-kd Ro, and anti-La autoantibodies were transported efficiently into the fetal circulation. Human IgG-apoptotic cell complexes were detected in the heart (atrial trabeculae and atrioventricular node), skin, liver, and newly forming bone of fetuses from mothers injected with anti-Ro/La sera but not control sera. The IgG binding was fetal-specific and organ-specific; transplacental autoantibodies did not bind to apoptotic cells in the fetal thymus, lung, brain, or gut. The complexes were not associated with an inflammatory reaction. Injection of mothers with affinity-purified anti-La autoantibodies (but not anti-Ro/La Ig depleted of anti-La) revealed an identical location of IgG binding to apoptotic cells in the fetuses. CONCLUSION: This is the first study to demonstrate that transplacental anti-La autoantibodies bind specifically to apoptotic cells in selected fetal organs in vivo, similar to the organ involvement in NLS. We hypothesize that additional factors are required to promote proinflammatory clearance of IgG-apoptotic cell complexes and subsequent tissue damage.

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 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.

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, Sjögren's 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.