Interaction of human chagasic IgG with human colon muscarinic acetylcholine receptor: molecular and functional evidence

Abstract

Background and aims: Gastrointestinal disorders is one of the clinical manifestations of chronic Chagas' disease. The pathogenesis seems to be associated with autonomic dysfunction. Here, we consider the muscarinic cholinoceptor mediated alteration in distal colon function in chagasic megacolon.

Patients: Patients were divided into four groups: group I, chronic chagasic patients with megacolon; group II, chronic chagasic patients without megacolon; group III, non-chagasic patients with megacolon; and group IV, normal healthy volunteers (control).

Methods: Binding assay and immunoblot of cholinoceptors from human and rat colon and enzyme immunoassay (ELISA) using a synthetic 24mer peptide corresponding to the second extracellular loop of human M2 muscarinic acetylcholine receptors (mAChR) were used to detect the presence of serum antibodies. The effect of antibodies on basal tone and 3',5'-cyclic monophosphate (cAMP) production of human and rat distal colon strips were also tested.

Results: Group I but not the other groups had circulating antibodies capable of interacting with human colon activating M2 mAChR, as they competed with binding of specific radioligand to mAChR and interacted with the second extracellular loop of human M2 mAChR. Moreover, affinity purified anti-M2 peptide IgG from group I, in common with monoclonal antihuman M2 mAChR, recognised bands with a molecular weight corresponding to colon mAChR. This antibody also displayed an agonist-like activity, increasing basal tone and decreasing cAMP accumulation. Both effects were blunted by AF-DX 116 and neutralised by the synthetic peptide.

Conclusions: In chagasic patients with megacolon there are antibodies that can recognise and activate M2 mAChR. The implications of these autoantibodies in the pathogenesis of chagasic megacolon is discussed.

Figure 1

Concentration dependent inhibition of [³H] N-methylscopolamine (NMS) binding on colonic smooth muscle membranes. Rat (A) and human (B) colon smooth muscle homogenates were incubated with serum IgG from either chagasic patients with megacolon or normal individuals in the presence of [³H] NMS. Control binding of 100% refers to the binding of the muscarinic radioligand to membranes treated with no IgG. Data are mean (SEM) of triplicate determinations of five IgGs from different chagasic or normal subjects.

Figure 2

Immunoreactivity of anti-muscarinic acetylcholine receptor (mAChR) antibodies from chagasic sera against the second extracellular loop of the human M₂ mAChR tested by ELISA. Microtitre wells were coated with 1 µg of M₂ peptide and an enzyme immunoassay was performed (see materials and methods). (A) Mean (SEM) optical density (OD) values, representing triplicate assays from 15 chagasic patients with megacolon, 17 chagasic patients without megacolon, and three non-chagasic patients with megacolon. (B) Mean (SEM) optical density (OD) values, representing triplicate determinations of five affinity purified antipeptide IgGs from different chagasic patients with megacolon in the presence (+) or absence (−) of 1×10⁻⁵ M peptide.

Figure 3

Immunoblotting of M₂ muscarinic acetylcholine receptor (mAChR) on rat (A) and human (B) colon smooth muscle homogenates. Lane 2: IgG fraction from chagasic patients with megacolon; lane 3: affinity purified antipeptide IgG from chagasic patients with megacolon; lane 1: monospecific mouse antihuman M₂ mAChR IgG; and lane 4: serum IgG fraction from normal patients.

Figure 4

Concentration dependent effect of the anti-M₂ peptide IgG fraction from chagasic patients with megacolon on rat (A) and human (B) colon tonic contractions. Isolated distal colonic strips were incubated with affinity purified antipeptide antibodies in the presence (+) or absence (−) of 1×10^-5 M peptide and tonic contractions were recorded. Values are mean (SEM) of five antipeptide IgG fractions in each group.

Figure 5

Original tracings showing dose dependent increases in human colon tonic contractions by anti-M₂ peptide IgG (IgG log [M]) from chagasic patients with megacolon alone (A) and after exposed tissues to 5×10⁻⁶ M AF-DX 116 (B), 5×10⁻⁵ M synthetic M₂ peptide (C), and 5×10⁻⁶ M pertussis toxin (D) over 30 minutes before anti-M₂ peptide IgG from chagasic patients with megacolon was added.