Agonistic Autoantibodies to the β2-Adrenergic Receptor Involved in the Pathogenesis of Open-Angle Glaucoma

Abstract

Glaucoma is a frequent ocular disease that may lead to blindness. Primary open-angle glaucoma (POAG) and ocular hypertension (OHT) are common diseases with increased intraocular pressure (IOP), which are mainly responsible for these disorders. Their pathogenesis is widely unknown. We screened the sera of patients with POAG and OHT for the prevalence of autoantibodies (AAb) against G protein-coupled receptors (GPCRs) in comparison to controls. Employing frequency modulation of spontaneously contracting neonatal rat cardiomyocytes in vitro, agonistic GPCR AAb were to be detected in roughly 75% of the patients with POAG and OHT, however, not in controls. Using inhibitory peptides the AAb' target was identified as β2 adrenergic receptor (β2AR). The AAb interact with the second extracellular loop of β2AR. The peptides 181-187 and 186-192 were identified as binding sites of the AAb within the extracellular loop II. The binding of the AAb to β2ARs was verified by surface-plasmon-resonance analysis. The isotype of the AAb was (immunoglobulin) IgG3. In an additional pilot principal-of-proof study, including four patients with POAG, the removal of the AAb against the β2AR and other immunoglobulins G by immunoadsorption resulted in a transient reduction of IOP. These findings might indicate a possible role of agonistic AAb directed against β2ARs in the dynamics of aqueous humor and might support a contribution of adaptive autoimmunity in the etiopathogenesis of POAG and OHT.

Trial registration: ClinicalTrials.gov NCT00494923.

Keywords: agonistic; autoantibodies; glaucoma; immunoadsorption; ocular hypertension; β2-adrenergic receptor.

Figure 1

Antibodies stimulating rat cardiomyocytes in sera of patients with POAG and OHT. Immunoglobulin-enriched serum fractions were added to cardiomyocyte cultures in a 1:20 dilution and incubated for 60 min at 37°C. The data are displayed as the changes of the beats/min. Each point represents 6–10 separate single-cell check points in a culture flask. Repeated measurements of the effects with different cell cultures prepared on different days resulted in almost identical data. The differences between patients with POAG or OHT versus healthy controls were highly significant. Primary open-angle glaucoma (POAG, n = 37); ocular hypertension (OHT, n = 9); cataract (n = 10); healthy donors (NHD, n = 10).

Figure 2

β2-adrenergic receptor (β2AR) agonistic autoantibodies (AAbs) in patients with primary open-angle glaucoma (POAG) or ocular hypertension (OHT). The neonatal cardiomyocytes were incubated with the immunoglobulin fractions from POAG (n = 33) and OHT (n = 14) patients as described in Figure 1. The agonistic effect of the AAbs was blocked by the β2AR antagonist ICI118551 (ICI; 0.1 μM). ICI (0.1 μM) did not affect the basal beating rate of the cardiomyocytes.

Figure 3

Time response curve of β2 adrenergic agonists. Effect of (A) the agonistic autoantibody (AAb) prepared from patients with primary open-angle glaucoma (POAG) or ocular hypertension (OHT) on spontaneously beating rat cardiomyocytes and (B) clenbuterol (Clen). Clenbuterol induced a desensitization of the β adrenergic response within 2 h. After 2 h, clenbuterol was washed out and restimulation with clenbuterol resulted in approximately 30% of the initial stimulation (B). β2-adrenergic receptor AAb showed no desensitization for at least 5 h (A). Before washing, the antibodies were removed from the receptors by ICI118551. After washing, the cells react to a clenbuterol stimulation with a maximal response (n = 10 for each point of measurement).

Figure 4

The agonistic anti-β2AR autoantibodies (AAbs) of patients with primary open-angle glaucoma (POAG) recognized the second extracellular loop of the β2-adrenergic receptor. The antibody preparations from patients with POAG (n = 5) were pretreated with loop-specific peptides corresponding to the extracellular loops I–III. The antibody–peptide complexes were added to the cardiomyocytes to measure remaining agonistic capabilities. The final AAb dilution was 1:40. The data are presented as the increase in beats/min versus baseline beating rates.

Figure 5

The agonistic anti-β2AR autoantibodies (AAbs) of patients with primary open-angle glaucoma (POAG) recognize the epitopes AINCYAN and ANETCCD of loop II. The procedure to detect epitope-specific responses of immunoglobulins isolated from POAG patients is similar to the one used in the studies for loop analyses. The final AAb dilution was 1:40. Data are displayed as individual measurements of five independent patients with POAG.

Figure 6

Agonistic anti-β2AR IgG autoantibodies in patients with primary open-angle glaucoma. The neutralization of IgG from the immunoglobulin-enriched fraction lead to the loss of the stimulatory potential. Antibodies against IgM had no effect. The β2 receptor agonist clenbuterol served as positive control (not shown).

Figure 7

IgG3 subclass of the agonistic anti-β2-adrenergic receptor autoantibodies in patients with primary open-angle glaucoma. The neutralization of IgG3 from the immunoglobulin-enriched fraction leads to the loss of the stimulatory potential. Antibodies against IgG1, 2, and 4 had no effect. The β2 receptor agonist clenbuterol served as positive control (not shown).

Figure 8

Time course of IOP, levels of autoantibodies (AAbs) and number of local antiglaucomatous eye drops during immunoadsorption (IA) for the patient with primary open-angle glaucoma (POAG). Patient 1/2/3/4 (A)/(B)/(C)/(D): a decrease in IOP under reduced number of local antiglaucomatous therapy was to be observed in all patients, going along with a decrease of the AAb. Intraocular pressure (IOP) increased, when AAbs relapsed after the IA; OD, right eye (black); OS, left eye (red); grayish area, data before IA; visit 1, IOP graph: less than 3 months before immunoadsorption under maximal combination therapy, AAB against β2AR graphs: before immunoadsorption; visit 2 – IOP graph: two days before immunoadsorption with system medications (acetazolamide), yet without topical medications, AAB against β2AR graphs: at the end of immunoadsorption; IOP/AAB against β2AR graphs: visit 3–2 weeks after immunoadsorption; visit 4–1 month after immunoadsorption; visit 5–2 months after immunoadsorption; visit 6–4 months after immunoadsorption; visit 7–6 months after immunoadsorption; visit 8–12 months after immunoadsorption.

Figure 8

Time course of IOP, levels of autoantibodies (AAbs) and number of local antiglaucomatous eye drops during immunoadsorption (IA) for the patient with primary open-angle glaucoma (POAG). Patient 1/2/3/4 (A)/(B)/(C)/(D): a decrease in IOP under reduced number of local antiglaucomatous therapy was to be observed in all patients, going along with a decrease of the AAb. Intraocular pressure (IOP) increased, when AAbs relapsed after the IA; OD, right eye (black); OS, left eye (red); grayish area, data before IA; visit 1, IOP graph: less than 3 months before immunoadsorption under maximal combination therapy, AAB against β2AR graphs: before immunoadsorption; visit 2 – IOP graph: two days before immunoadsorption with system medications (acetazolamide), yet without topical medications, AAB against β2AR graphs: at the end of immunoadsorption; IOP/AAB against β2AR graphs: visit 3–2 weeks after immunoadsorption; visit 4–1 month after immunoadsorption; visit 5–2 months after immunoadsorption; visit 6–4 months after immunoadsorption; visit 7–6 months after immunoadsorption; visit 8–12 months after immunoadsorption.