(Received for publication, December 21, 1996, and in revised form, March 10, 1997)
From the Centro de Estudios Farmacologicos y
Botanicos, Consejo Nacional de Investigaciones Cientificas y
Tecnicas, Buenos Aires 1414, Argentina and the ¶ Department of
Molecular Pharmacology and Biological Chemistry, Northwestern
University Medical School, Chicago, Illinois 60611
Chronic Chagas' disease is associated with pathologic changes of the cardiovascular, digestive, and autonomic nervous system, culminating in autonomic denervation and congestive heart failure. Previously, circulating autoantibodies that activate signaling by cardiac muscarinic acetylcholine receptors (mAChRs) have been described. However, it remains unclear whether the chagasic IgGs directly interact with the m2 mAChRs (predominant cardiac subtype), and, if so, whether chronic exposure of the mAChRs to such activating IgGs would result in receptor desensitization. Here we performed studies with purified and reconstituted hm2 mAChRs and demonstrate that IgGs from chagasic serum immunoprecipitated the mAChRs in a manner similar to an anti-m2 mAChR monoclonal antibody tested in parallel. The chagasic antibodies did not directly interact with the ligand binding site, because the binding of radiolabeled antagonist was unchanged by the addition of the chagasic IgG. In intact cells stably expressing the hm2 mAChR, the chagasic IgGs, but not normal IgGs, mimicked the ability of the agonist acetylcholine to induce two effects associated with agonist-induced receptor desensitization: a decrease in affinity for agonist binding to m2 mAChR and sequestration of the hm2 mAChRs from the cell surface. The results demonstrate that the chagasic IgGs can directly interact with and desensitize m2 mAChRs and provide support for the hypothesis of autoimmune mechanisms having a role in the pathogenesis of Chagas' cardioneuromyopathy.
Chagas' disease, one of the most common determinants of
congestive heart failure and sudden death in the world, is transmitted by the parasite Trypanosoma cruzi (1, 2). Most patients survive the acute phase of the disease asymptomatically; the chronic stage of the disease develops over 20-30 years and is manifested by
cardiovascular, digestive, and autonomic nervous system disorders (3-6). Several studies have focused on the disease-related loss of
function of the autonomic receptors, the -adrenergic and muscarinic cholinergic receptors (mAChR)1 (5-11). The
paradoxical severe involvement of the heart in the absence of any form
of the parasite there has prompted proposals that autoimmune mechanisms
participate in the pathogenesis of this cardiac neuromyopathy (12-14).
Chagasic patients have been found to possess circulating antibodies
that are able to interact with and modulate the activity of cardiac
-adrenergic and mAChRs (15-19). This report focuses on the
molecular events initiated by the interaction of the chagasic
antibodies with the mAChRs to begin to understand how the antibodies
could ultimately account for the abnormalities of cardiac autonomic
function characteristic of the late stages of Chagas' disease.
Previously, chagasic antibodies were shown to mimic agonists of mAChR
to cause inhibition of cardiac contractility, stimulation of cGMP
levels, and attenuation of cAMP synthesis (18, 19). Thus, the in
vivo situation during the course of Chagas' disease could be
similar to that of a persistent stimulus, i.e. circulating
antibodies that activate cardiac mAChRs could result in receptor
desensitization. Such a scenario could cause receptor/G-protein
uncoupling and loss of receptors from the cell surface and explain the
progressive blockade of receptors that is observed in the disease (20,
21). To test this hypothesis, we used both purified human m2 mAChRs and
mammalian CHO cells transfected with human m2 mAChRs to assess the
ability of chagasic IgG to directly interact with and desensitize the
mAChR. The results support a role for the chagasic antibodies in
causing loss of mAChR function.
Trypanosoma
cruzi-infected patients from metropolitan Buenos Aires were
studied. These patients, classified as Group I based on WHO Expert
Committee on Chagas' disease criteria (1), had positive T. cruzi serology and the absence of clinical symptoms with normal
electrocardiogram and chest x-ray. Among this group, sera were chosen
for their previously demonstrated ability to activate rat atrial mAChR
in an atropine-sensitive manner (18, 19). Normal sera taken as controls
were from volunteers who had negative serology for Chagas' disease,
presented no evidence of cardiovascular or chronic systemic disease or
acute viral or febrile disease, and had normal electrocardiogram and
chest x-ray. The age of patients and controls ranged between 25 and 50 years. IgG was isolated from chagasic or normal sera by means of
DEAE-cellulose chromatography (Bio-Rad) as shown (18) and tested for
purity by immunoelectrophoresis with goat anti-whole serum or anti-IgG antiserum (Sigma). The IgG concentration was determined by
radioimmunodifussion assay. F(ab)2 fragments were obtained
from normal or chagasic patients by pepsin digestion of IgG fractions
in 0.1 M sodium acetate buffer (pH 4.5) for 22 h at
37 °C, dialysis against phosphate-buffered saline (PBS), and
chromatography on Sephadex G-200. Purification of IgG was assessed by
SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis.
Human mAChRs (hm2 mAChR) from Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus encoding the human m2 mAChR were purified and reconstituted as described (22-24). The hm2 mAChR purified from Sf9 cells is expressed as a 55-kDa protein, which is smaller than that observed in mammalian cells because it lacks the extensive glycosylation associated with the mammalian expressed receptor (24). Recombinant virus was kindly provided by Drs. Eric M. Parker and Elliot Ross (University of Texas, Dallas, TX).
Culture of m2 Chinese Ovary CellsChinese hamster ovary cells stably transfected with hm2 mAChRs (m2 CHO cells; Ref. 25) were kindly provided by Dr. Ernest Peralta (Harvard University, Cambridge, MA). Cells were grown in Dulbecco's modified Eagle's medium:Ham's F-12 (DMEM:F-12) supplemented with 10% dialyzed fetal bovine serum, 100 units/ml of penicillin and streptomycin, and 2 µM glutamine in the presence of 250 nM methotrexate. Cells were plated in 60- or 100-mm dishes and used at about 80% confluency. The density of mAChRs per cell was determined by saturation binding of [3H]QNB to intact cells (26).
Immunoprecipitation AssaysPurified reconstituted hm2 mAChRs were immunoprecipitated with chagasic antibodies or a monoclonal anti-m2 antibody (27) generously provided by Dr. M. Schimerlik from Oregon State University (Corvallis, OR). Chagasic or normal sera or anti-m2 hybridoma supernatant were precoupled to protein A-agarose beads (50 µl, Pierce) overnight at 4 °C. The beads were then incubated with ~0.7 pmol of reconstituted mAChRs at 4 °C for 5 h and washed thoroughly. The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis using 8.5% gels and then transferred to nitrocellulose membranes. Proteins were visualized by means of the anti-m2 monoclonal antibody and a goat anti-mouse IgG conjugated to peroxidase followed by the ECL reaction (Amersham Corp.) as described (26).
Desensitization and Sequestration of the mAChRThe m2 CHO
cells were grown in 100-mm dishes, washed with PBS, and then allowed to
stabilize for 10 min at 37 °C with serum-free fresh DMEM:F-12
containing 10 µM eserine (a cholinesterase inhibitor) before the addition of treatments to induce desensitization or sequestration. To assess the effects of chagasic sera to cause decrease
in high affinity agonist binding, cells were treated with serum-free
medium containing either PBS (as a no drug/no IgG control), the agonist
acetylcholine, or chagasic or normal IgGs. Cells were incubated for
1 h at 37 °C in a 5% CO2 atmosphere with one of
the above reagents, and after extensive washing, they were harvested in
10 mM phosphate buffer (pH 7.4), 1 mM EDTA, 0.25 M sucrose. All steps were performed at 4 °C. Cells
were centrifuged at 400 × g for 15 min, and crude
membranes were prepared as described previously (27). To test for
effects of pretreatments with agonist or IgGs on the affinity of the
receptors for agonist, the agonist carbachol was used to compete for
[3H]QNB binding to mAChR in the membrane preparations.
Membranes were incubated at 37 °C for 75 min in 20 mM
phosphate buffer (pH 7.4), 1 mM EDTA, 2 mM
MgCl2, [3H]QNB (0.5-0.6 nM), and
varying concentrations of carbachol in the presence or the absence of
the guanine nucleotide analogue 5-guanylylimidodiphosphate (Gpp(NH)p,
at 100 µM). The data were analyzed with the curve-fitting
program LIGAND (28).
To assess for sequestration of hm2 mAChRs, experiments were performed on intact m2 CHO cells using the hydrophilic ligand [3H]N-methylscopolamine (NMS) to determine changes in cell surface m2 mAChR number, whereas the hydrophobic ligand [3H]QNB was used to assess total receptors. After pretreatment with drugs or IgGs as described above, the culture dishes were placed on ice, and the cells were thoroughly washed with cold PBS. Then they were spun down at 400 × g, resuspended in ice-cold DMEM:F-12 and assessed for viability by trypan blue exclusion, and suspensions with more than 95% viable cells were used for binding assays. Saturating concentrations of [3H]NMS or [3H]QNB were incubated with cells in Hepes-buffered DMEM:F-12 in the presence or the absence of 1 µM atropine to calculate specific binding for 2 h at 4 °C for [3H]NMS and for 75 min at 37 °C for [3H]QNB. Cells were filtered onto GF/C glass fiber filters, and radioactivity was counted in a Beckman spectrometer. Control levels of surface and total receptors were defined as the levels in cells treated the same as above but with the corresponding volume of PBS instead of drug or IgG. These values were considered as 0% internalization of each assay. The percentage of internalization due to drug or IgG treatments was calculated as the percentage of loss of surface receptors measured with [3H]NMS in treated samples compared with 0% internalization controls. Down-regulation was defined as a net decrease in the total receptor pool as assessed by [3H]QNB binding assays. Variations in cell density per plate were controlled by normalizing all data on the basis of mg protein/plate.
To test the ability of chagasic antibodies to directly
interact with purified hm2 mAChRs, immunoprecipitation experiments were
performed with several chagasic or normal sera and purified hm2 mAChRs
reconstituted in vesicles. As a control, an equivalent amount of the
hm2 mAChR were also immunoprecipitated with a known monoclonal anti-m2
mAChR antibody (26). The immunoprecipitates were fractionated by
SDS-polyacrylamide gel electrophoresis, and immunoprecipitated mAChR
was visualized by immunoblotting with the anti-m2 mAChR monoclonal
antibody (Fig. 1). The results demonstrated that the
chagasic sera immunoprecipitated the hm2 mAChR (Fig. 1, lanes
1 and 2). Two immunoreactive bands were detected by the anti-m2 mAChR mAb, one corresponding to a protein of 55 kDa, the expected size for the hm2 mAChR purified from Sf9 cells, and a second
species at ~116 kDa that likely represented the receptor dimer (24).
The immunoreactive bands that were present in the immunoprecipitates
from the chagasic sera migrated identically to those immunoprecipitated
by the m2 mAb (Fig. 1, lane 5). The amount of mAChR
immunoprecipitated by the chagasic antisera was lower than the fraction
of receptors immunoprecipitated by the m2 mAb run in parallel; however,
the amount of mAChR immunoprecipitated by the chagasic sera was
significant and far greater than the trace amounts (background levels)
immunoprecipitated by the normal sera used as control (Fig. 1,
lanes 3 and 4). These results provide a direct
demonstration of the recognition of the hm2 mAChR by antibodies present
in chagasic sera.
To test whether the chagasic sera interacted directly with the ligand binding site of the m2 mAChR or with another domain, we tested whether the chagasic sera could compete with the antagonist [3H]QNB in ligand binding assays. For these studies we prepared membranes from CHO cells expressing the hm2 mAChR and performed saturation binding isotherms in untreated membranes and membranes treated with either normal or chagasic IgG. No differences in the Kd or Bmax values were observed among the three groups (Table I).
|
The possibility that the persistent
activation of m2 mAChRs by chagasic antibodies could lead to receptor
desensitization was evaluated. Desensitization of G-protein-coupled
receptors is associated with several events, in particular,
receptor/G-protein uncoupling and sequestration of the receptors away
from their normal membrane environment. A hallmark of receptor
uncoupling from G-proteins is the conversion of receptors from a high
to low affinity state. The high affinity state for agonists is
considered to be a receptor/G-protein complex, whereas the lower
affinity state is thought to represent the free receptor. In agonist
binding studies in vitro a loss of high affinity agonist
binding can be induced upon the addition of GTP or its analogs,
presumably due to the activation of the G-protein and its subsequent
dissociation of the G-protein from the receptor. Agonist-induced
desensitization of the -adrenergic receptor has been associated with
the loss of high affinity agonist binding and conversion of receptors
to a low affinity state (29).
To test for desensitizing effects of chagasic antibodies on agonist
binding properties, we compared the effects of pretreatment of intact
cells with either the agonist acetylcholine or normal or chagasic IgG
on agonist affinity. After each pretreatment, membranes were prepared
and used in carbachol/[3H]QNB competition binding
experiments in the presence or the absence of a guanine nucleotide
analogue. In assays of membranes from control cells (treated with PBS),
shallow, biphasic agonist competition curves were obtained in the
absence of Gpp(NH)p, reflecting both high and low affinity agonist
binding (Fig. 2a). The addition of Gpp(NH)p
right shifted and steepened the curves, as expected for the guanine
nucleotide-dependent conversion of high affinity sites into
low affinity sites (Fig. 2a, untreated). The
corresponding IC50 values and Hill coefficients reflected
these changes (Table II). In addition, analysis of the
data with the curve fitting program LIGAND (28) demonstrated the
expected results, in that a high and low affinity state were observed
and Gpp(NH)p caused a conversion of receptors from the high to low
affinity state (Table III). Pretreatment of cells either
with the agonist acetylcholine under conditions that lead to
desensitization (27) or with chagasic IgG produced rightward shifts of
the concentration-displacement curves of carbachol in the absence of
Gpp(NH)p compared with untreated cells (compare Fig. 2, b
and d, to Fig. 2a; Table II). The effects caused
by either pretreatment were not as extensive as those induced by the
addition of Gpp(NH)p to the in vitro assays. The protease digestion fragment of chagasic IgG, F(ab)2, had similar
effects to the nondigested chagasic IgG (data not shown). The shift
induced by chagasic IgG was not observed when incubations were
performed with normal IgG (Fig. 2c, Table II). Analysis of
the data with the LIGAND program indicated that the decrease in overall
affinity caused by the chagasic IgG was due to an increase in the value of KH, whereas there was no detectable change in
the percentage of receptors in the higher affinity state (Table III).
Treatment of the cells with agonist produced a similar trend (Table
III). The LIGAND analysis also suggested that the pretreatment of the cells by either agonist or chagasic IgG produced effects
distinguishable from guanine nucleotides, because the percentage of
receptors in the high affinity state was decreased by the addition of
Gpp(NH)p to the in vitro assays (Table III). The data
obtained with the normal IgG were similar to those from the untreated
cells (Table III).
|
|
It is not entirely clear why the effects of the pretreatments with
agonist or chagasic IgG produced increases in KH
without the expected decrease in RH. One
explanation is that the chagasic IgG or agonist only partially
desensitized the receptor. Indeed, in studies of the desensitization of
the -adrenergic receptor, similar results were obtained (29). With
partial desensitization, a decrease in numbers of high affinity
-adrenergic receptors was less apparent, and the main effect seemed
to be a small increase in KH (29) as was
observed here. The present results also were similar to those we
previously obtained in chick heart tissue treated with desensitizing
doses of acetylcholine, where KH for agonist
binding to the mAChR was increased, whereas the percentage of high
affinity receptors was less affected (30). In addition, we observed
very similar trends in studies with heterologously expressed hm2 mAChR
in Sf9 cells (31). It appears that the effects of desensitization on
the decreased affinity of agonist binding to the m2 mAChR are less
extensive than can be induced in vitro upon the addition of
guanine nucleotides to ligand binding assays. Further studies will be
necessary to dissect these complex patterns of responses that are
elicited by desensitization. Nevertheless, the present results are
consistent with the conclusion that acute exposure of the m2 CHO cells
to the chagasic IgG resulted in a decreased affinity of the receptor
for agonist. These desensitizing effects of the chagasic IgG were
similar to those induced by exposure to agonist. The results are
consistent with the suggestion that the agonist-like activity of the
chagasic sera can induce desensitization of the m2 mAChR in intact
cells.
To test if
chagasic IgG induced sequestration of receptors, cells expressing m2
mAChRs were incubated for different times with increasing
concentrations of chagasic or normal IgGs or F(ab)2 before
binding assays were performed. Comparable incubations with acetylcholine were run in parallel. The hydrophilic ligand
[3H]NMS and the lipophilic ligand [3H]QNB
were used in whole cell binding assays to assess cell surface and total
receptors, respectively. Both the chagasic IgG and acetylcholine induced a time-dependent sequestration of the mAChRs to a
similar extent, whereas normal IgG had no significant effect (Fig.
3a). The effects of both acetylcholine and
the chagasic IgG were concentration-dependent; the maximum
effect observed with the chagasic IgG occurred at 0.2 mg/ml and was
similar to that caused by 100 nM acetylcholine (Fig.
3b). The chagasic F(ab
)2 also induced
internalization but with a lower efficacy (Fig. 3b). Normal
IgG was ineffective at any concentration (Fig. 3b). In the
time periods tested, no down-regulation of receptors by chagasic IgGs,
normal IgGs, or acetylcholine was observed based on measurements of
total mAChR with [3H]QNB (data not shown). The present
report provides evidence of a specific interaction between antibodies
from the serum of chagasic patients and hm2 mAChRs and describes some
early consequences of the persistent activation of the receptors by the
antibodies. The results obtained support the hypothesis that antibodies
having an "agonist-like" activity can participate in the regulation
of the receptors they bind to, thus influencing their normal function in vivo. The antibody-induced decrease in agonist affinity
occurred after 1 h of incubation of m2 CHO cells with chagasic
IgGs or F(ab
)2, whereas internalization was extensive by
90-120 min, similar to the effect of acetylcholine. The data
demonstrate that the agonist-like behavior of the antibodies is
sufficient to induce the series of reactions associated with
desensitization of the receptors. The results are consistent with our
previous observations on the ability of chagasic IgG to impair
agonist-induced inhibition of contractility of rat atria after
prolonged incubation with IgGs (18). It is unlikely that the present
results could have arisen from nonspecific interactions, because the
data in Fig. 1 demonstrated an ability of the chagasic antibodies to
directly interact with the m2 mAChR. In addition, whereas previous
studies have suggested effects of chagasic serum on adrenergic
signaling (15-19), the CHO cells used in this study lack adrenergic
receptors, making it unlikely that the observed desensitizing effects
of the chagasic antibodies on the mAChR were due to cross-talk arising from effects on signaling pathways downstream of the receptors.
Chagasic patients exhibit several signs of impaired parasympathetic function (14); however, the molecular mechanisms involved remain unexplained. Thus, the major point of our report is that early events of agonist-promoted desensitization of m2 mAChR can be initiated by autoantibodies that bind to and persistently activate the receptors. Further studies will be required to address the issue of whether early molecular alterations in the function of the cardiac autonomic nervous system during Chagas' disease are related to the presence of autoantibodies promoting receptor desensitization. Interestingly, the existence of autoantibodies against autonomic receptors has been reported in other cardiomyopathies such as human dilated cardiomyopathy (32-34) and congenital heart block (35), but evidence is still lacking about the mechanisms underlying their potential pathological role. Whether or not autoantibodies play a general role in other cardiomyopathies remains to be determined.
We thank Judy Ptasienski for purification and reconstitution of the m2 mAChR.