Autoantibodies Blocking M3 Muscarinic Receptors Cause Postganglionic Cholinergic Dysautonomia

Abstract

A 10-year-old girl presented with ileus, urinary retention, dry mouth, lack of tears, fixed dilated pupils, and diffuse anhidrosis 7 days after a febrile illness. We hypothesized that her syndrome was due to autoimmunity against muscarinic acetylcholine receptors, blocking their activation. Using an indirect enzyme-linked immunosorbent assay for all 5 muscarinic receptors (M₁ -M₅ ), we identified in the patient's serum antibodies that selectively bound to M₃ receptors. In vitro functional studies confirmed that these autoantibodies selectively blocked M₃ receptor activation. Thus, autoantibodies against M₃ acetylcholine receptors cause acute postganglionic cholinergic dysautonomia. ANN NEUROL 2020;88:1237-1243.

Figure 1:. Autonomic and somatic efferent neurons and target organs.

Nicotinic acetylcholine receptors (nAchR) are located in the sympathetic and parasympathetic ganglia and in striatal muscle. Muscarinic acetylcholine (ACho) receptors (mAchR) are located in sweat glands and organs and glands throughout the body (sinoatrial node in the heart, gastrointestinal system, detrusor muscle of the bladder, salivary glands, lacrimal glands, pupillary constrictor muscles, and pancreas). Given the lack of orthostatic hypotension and muscle weakness, the phenotype of postganglionic cholinergic dysautonomia is consistent with selective muscarinic failure, sparing nicotinic acetylcholine and α-adrenergic (α-AR) receptors. NE: norepinephrine.

Figure 2.. Oral and ocular manifestations of selective muscarinic failure.

A. Extremely dry tongue during the acute phase. B. Upon discharge her tongue had improved but still remained significantly dry. C. At the 6-month follow-up visit, her tongue had normal appearance. D. During the acute phase, pupils were mydriatic and non-reactive to light (Video) and lacrimation was absent (Schirmer test < 5 mm/5 min). E. To asses for the presence of parasympathetic denervation supersensitivity, we evaluated pupillary responses to pilocarpine instillation 0.125%. After pilocarpine 0.125 % both pupils constricted tonically (2-mm diameter) remaining miotic for 4-hours, consistent with parasympathetic denervation supersensitivity.

Figure 3.. Patient’s serum from the acute phase contains antibodies blocking M₃ muscarinic receptors.

(A) ELISA results after addition of the patient’s sera. We detected a significant signal when the patient’s serum during the acute phase was exposed to cells expressing cholinergic M₃ muscarinic receptors (P<0.01 vs. healthy control serum), but no difference when the serum was exposed to cells expressing M₁, M₂, M₄ or M₅ muscarinic receptors (n.s. vs. healthy control serum, not indicated in the figure). This signal was significantly reduced to levels similar to those observed when we used serum from an age- and sex-matched health control when we used the patient’s 6-month follow-up visit (6-m) serum (P<0.05 compared to the acute phase; and P=n.s. compared to the healthy control) Data are mean±SEM of three independent experiments. *P<0.05; **P<0.01; One-Way ANOVA. n.s.=not significant. (B) ELISA results after addition of a commercially available anti-M₃ antibody. As expected, we detected a significant binding only in cells expressing M₃ receptors, but not in the cells expressing the other muscarinic receptors, or in CHO cells devoid of muscarinic receptors (CHO only) when we used a commercial, specific anti-M₃ antibody. Data are mean ± SEM of 3 independent experiments. ****P<0.0001; One-Way ANOVA. (C) ELISA results comparing anti-M₃ antibody binding to cell expressing M₃ receptors in the presence or absence of the patient’s serum. Both the commercially available anti-M₃ antibody (anti-M₃ Ab) and the patient’s serum during the acute phase (Acute Phase) showed similar binding activity to the M₃ cells (P=n.s.), with higher signal compared to the sample from the healthy control (Con.) and the patient’s serum at the 6-month follow-up visit (6-m Visit). This supports the presence of specific, biologically active, anti-M₃ receptors in the patient’s serum during the acute phase. Data are mean ± SEM of 3 independent experiments. *P<0.05; ****P<0.0001; One-Way ANOVA. n.s.=not significant. (D) [³⁵S]GTPγS binding assay. The [³⁵S]GTPγS binding assay showed blockade of M₃ receptor activation induced by increasing concentrations of pilocarpine, a known M₃ cholinergic agonist, in the presence of serum from the patient (Acute Phase) compared to serum obtained from a healthy control (P<0.001, acute phase vs. control). The activation of the M₃ receptor was less impaired during the 6-month follow-up visit (6-m Visit), but still significantly inferior to the control serum (P<0.05 6-month vs. control). **P<0.01; ***P<0.001 One-Way ANOVA. n.s.=not significant. (E) Panel E shows data in panel D normalized (percentage) to the corresponding basal values. Data are mean±SEM of 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001; One-Way ANOVA. n.s.=not significant. (F) Intracellular Ca⁺² mobilization assay with 1 μM pilocarpine. In the presence of 1 μM pilocarpine, a known muscarinic agonist, the addition of the patient’s serum during the acute phase elicited a response significantly blunted compared to that observed in the presence of the healthy control serum (Con.) (P<0.001 vs. Con.). The response in the presence of the patient’s serum obtained at the 6-month follow-up visit (6-m Visit) was more intense than during the acute phase (P<0.001), and similar to that observed in the presence of the healthy control’s serum (P=.n.s.). Data are mean±SEM of three independent experiments. **P<0.01; ***P<0.001; n.s.=not significant. (G) Intracellular Ca⁺² mobilization assay with increasing concentrations of pilocarpine. In the presence of the patient’s serum (Acute Phase) the relative fluorescence at increasing concentrations of pilocarpine was significantly reduced compared to the response observed in the presence of the healthy control’s serum (P<0.01 with pilocarpine at 0.1 μM and 1 μM; and P<0.001 with pilocarpine 10 μM), indicating lack of M₃ receptor activation. When serum from the patient at 6-month follow-up visit (6-m Visit) was used, the relative fluorescence was significantly increased, and became similar to the values observed in the presence of the healthy control’s serum. Data are mean±SEM of three independent experiments. **P<0.01; ***P<0.001.