(The FASEB Journal. 1999;13:2015-2020.)
© 1999 FASEB
Presence of antibodies against the third intracellular loop of the m2 muscarinic receptor in the sera of chronic chagasic patients
FERNANDA COUTINHO RETONDARO*,
PATRICIA C. DOS SANTOS COSTA,
ROBERTO COURY PEDROSA
and
ELEONORA KURTENBACH*1
* Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas,
Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Cidade Universitária, 21941–900, Rio de Janeiro, Brazil
1Correspondence: Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Cidade Universitária, 21941–900, RJ, Brazil. E-mail: kurten{at}bioqmed.ufrj.br
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ABSTRACT
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Patients in the chronic phase of Chagas' disease suffer from a slowly
evolving inflammatory cardiomyopathy that can lead to severe cardiac
dilatation, congestive heart failure, and death. This process appears
to be caused by autoimmune recognition of heart tissue by a mononuclear
cell infiltrate decades after infection with the parasite
Trypanosoma cruzi. Recent evidence suggests that there
are circulating antibodies in chronic chagasic patients that alter the
physiological behavior of the heart on binding to G-protein-coupled
cardiovascular receptors, including ß1-adrenergic and m2 muscarinic
receptors. A 42 kDa fusion protein was constructed that contains the
central part of the third intracellular loop (i3;
Arg267-Arg381) of the human m2 muscarinic
receptor, linked to glutathione S-transferase. This fusion protein was
overexpressed in Escherichia coli and subsequently
purified by affinity chromatography. Based on Western blots, the i3
loop is specifically recognized by the sera of chronic chagasic
patients who have reached advanced stages of cardiac failure (according
to the Los Andes classification). Analysis of the prevalence and
distribution of these antibodies shows a strong association between
seropositive patients and moderate (group II) to severe (group III)
heart dysfunction.—Retondaro, F. C., dos Santos Costa, P. C., Pedrosa, R. C., Kurtenbach, E. Presence of antibodies against
the third intracellular loop of the m2 muscarinic receptor in the sera
of chronic chagasic patients.
Key Words: Trypanosoma cruzi • autoimmune disease • chagasic cardiomyopathy • G-protein-coupled receptors
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INTRODUCTION
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CHAGAS' DISEASE IS caused by the protozoan parasite
Trypanosoma cruzi and is endemic in many countries of South
America, where an estimated 16–18 million people are infected and 9
million people run significant risk of infection.
One of the most frequent clinical manifestation of chronic Chagas'
disease patients is the development of `panmyocarditis', defined as
myocardial damage associated with mononuclear inflammatory foci
scattered throughout the heart (1)
. The coexistence of
areas of myositic degeneration, inflammatory infiltration, and fibrosis
suggests a chronic evolving process. The cardiopathy usually occurs in
25–30% of infected individuals 20–30 years after T. cruzi
primary infection, leading to heart failure and sudden death. The
remaining of T. cruzi-infected individuals either remain
asymptomatic (50–60%) or develop denervation of parietal smooth
muscle in the digestive tract (5–10%) (1)
Since the intracellular forms of the parasite T.
cruzi, which causes the disease, are rarely found in chagasic
myocarditis, it has been proposed that the pathology reflects an
autoimmune process, possibly involving antigenic mimicry between
T. cruzi and heart antigens (2)
. This
hypothesis is reinforced by the finding that CD4+
T cells from a murine model of chronic Chagas' disease cardiomyopathy
can evoke similar heart lesions in naive, uninfected mice
(3)
.
It has been shown that targets of the autoimmune attack include the
G-protein-coupled receptors of the heart cell membrane such as the
ß1-adrenoreceptors and the m2 muscarinic cholinergic receptors. The
second extracellular loop of these receptors (o2) was the first to be
indicated as an autoimmune epitope in patients with idiopathic dilated
cardiomyopathy (4
5
6)
and Chagas' disease cardiomyopathy
(7
, 8)
. These authors suggested that negatively charged
amino acid residues in the second extracellular loop of G-coupled
receptors may mimic one of the immunodominant ribosomal proteins (P0)
of the parasite. As a result of the interaction of chagasic
immunoglobulin G (IgG) with muscarinic cholinergic receptor, Goin et
al. (9
, 10)
observed a decrease in atrial contractility
and cAMP formation.
Using another approach, Oliveira et al. (11)
recently
demonstrated that sera from chronic chagasic patients with complex
cardiac arrhythmias can reduce cardiac rate and induce AV conduction
blockade in isolated adult rabbit hearts perfused by Langendorff's
method. These effects appear to be mediated by antibodies interacting
with muscarinic receptors since they are partially abolished by the
muscarinic antagonist atropine.
The third intracellular loop (i3) of muscarinic cholinergic
receptors is important in conferring specificity for G-protein
coupling, directing the flow of information to effector molecules. In
addition, i3 is a target for kinase phosphorylation, which leads to
receptor desensitization (12
, 13)
. The i3 loop contains a
large proportion of negatively charged residues (75% of all negatively
charged residues in the entire receptor); compared to o2, it has a
larger percentage of residues that are identical with those of the
T. cruzi P0 protein (28.5% to i3 compared with 10% to o2).
These considerations led us to test the hypothesis that i3 could be a
target for autoimmune attack in chronic chagasic patients. A large
amount of a 42 kDa fusion protein comprising the central part of the
third intracellular loop of the human muscarinic subtype m2
(Arg267 to Arg381)
(i3m2AChR) plus the enzyme glutathione S-transferase from
Schistosoma japonicum was produced in Escherichia
coli. This fusion protein was used as antigen in Western blots to
test the sera of chagasic patients for the capacity to recognize i3.
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MATERIALS AND METHODS
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Chagas' disease patients
Sera were obtained from chagasic patients in Rio de Janeiro.
Most were originally from endemic areas in Minas Gerais and Bahia,
Brazil, and all had lived outside endemic areas for more than 20 years.
The existence of T. cruzi infection was confirmed by
complement fixation, passive hemagglutination, cruzipain-enzyme-linked
immunoassay (cruzipain-ELISA), and indirect immunofluorescence. These
patients were evaluated at the Department of Cardiology (Hospital
Universitário Clementino Fraga Filho, Rio de Janeiro) every 2
months. At each visit they underwent clinical evaluation and, when
necessary, laboratory trials including 12-lead ECGs, M-mode and
2-dimensional echocardiographies, 24-dynamic ECGs, and exercise stress
testing in addition to a complete biochemical evaluation, including
thyroid function assays. Patients with concomitant arterial
hypertension, chronic obstructive pulmonary disease, cardiomyopathy of
any origin other than Chagas' disease, valvular heart disease,
congenital cardiomyopathy, obstructive coronary disease, thyroid
dysfunction, excessive alcohol consumption, and known immunological and
systemic diseases were excluded.
Three groups of patients were selected as representative of distinct
stages of myocardial damage according to the modified Los Andes
classification (14)
: group I (group IA + IB)
(n=10), group II (n=5), and group III
(n=10). The criteria for these groups are summarized in
Fig. 1
. The control group (n=6) was composed of patients who were
hospitalized for orthopedic surgery and presented a negative response
for Chagas' serology, with normal cardiovascular function. Table 1
provides information about clinical conditions of all experimental
groups.
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Figure 1. Los Andes classification. ECG, electrocardiography; CHF, congestive
heart failure; Echo/Ventricular, ventricular echocardiography.
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Construction of recombinant plasmids and expression of the m2
mAChR fusion protein
A BamHI/SmaI 340 bp cDNA fragment
corresponding to the central part of the third intracellular loop
(Arg267-Arg381) of the
human m2AChR cDNA cloned into pCD (kindly provided by Dr. E. C.
Hulme, NIMR, London) was ligated into SmaI-digested,
phosphatased vector pGEX-3X (Pharmacia, Piscataway, N.J.). The pGEX-3X
expression vector was constructed to give a fusion polypeptide with the
carboxyl terminus of the 27.5 kDa glutathione S-transferase (GST) of
Schistosoma japonicum (15)
. Recombinant plasmid
(pGEX-3X-i3m2) was verified to be in frame by dideoxynucleotide
sequencing using T7 DNA polymerase (Pharmacia).
GST-i3m2 fusion protein was expressed in the DH5
strain of E.
coli using isopropylthiogalactoside (IPTG) to induce
transcription. Briefly, individual colonies were inoculated into 2 ml
of Luria Broth (LB) media containing 100 µg/ml ampicillin and
cultured overnight at 37°C. After a 150-fold dilution in LB, the
suspension was incubated in a rotatory shaker at 37°C until
OD600nm = 0.6 was reached. IPTG was then added to
a final concentration of 0.5 mM and the culture was incubated for an
additional 4 h. Cells were harvested by centrifugation at
2560 x g for 15 min at 4°C and the pellet was
suspended in 3 ml of MTPBS buffer (150 mM NaCl, 16 mM
Na2HPO4, and 4 mM
NaH2PO4 pH 7.3). The sample
was sonicated six times for 1 min in a Cole Parmer sonicator (output
5.0) and solubilized with Triton X-100 (1%, v/v). After centrifugation
at 5,000 x g for 10 min at 4°C, 1 ml of the
solubilized fusion protein (supernatant) was mixed with 3 ml of
glutathione Sepharose 4B (Pharmacia) and incubated for 2 h at
4°C in phosphate-buffered saline buffer (140 mM NaCl, 27 mM KCl, 10
mM Na2HPO4, 1.8 mM
KH2PO4 pH 7.2). The
Sepharose beads were washed 10 times with 6 ml of the same buffer and
the fusion protein (Fig. 2
A) was eluted by competition with 20 ml of 50 mM Tris-Cl (pH
8.0) containing 10 mM reduced glutathione. Bacteria containing pGEX-3X
but lacking the cDNA insert were used as a negative expression control.
Antibody production
Two rabbits were immunized with polyacrylamide-stained KCl gel
bands corresponding to the 42 kDa fusion protein (250 µg) emulsified
in incomplete Freund's adjuvant and injected subcutaneously at
multiple sites in the animal's back. This procedure was repeated 3 wk
later. The rabbits were first bled 1 wk after the second injection. An
intramuscular booster injection was given after 1 month. The antisera
obtained had midpoint titrations in an ELISA at a dilution of 1:5000
when the purified fusion protein was used as antigen. In Western blots,
the antibodies recognize the fusion protein and m2AChR expressed in CHO
cells (Fig. 2B
).
Western blots
The 42 kDa fusion protein GST-i3m2 (10 µg), membranes from CHO
cells transfected with human m2AChR (50 µg) (Research Biochemicals
International) and membranes from SF9 cells transfected with
rat m1AChR (50 µg) recombinant plasmid pBB2rm1AChR were used as
antigens for immunoblotting studies. Samples were subjected to
electrophoresis on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) (10% or 15% acrylamide) and
electrotransferred to PVDF membranes. After saturation with 5% (w/v)
milk defatted in TBST buffer (25 mM Tris-Cl, 140 mM NaCl, 27 mM KCl,
0.05% (v/v) Tween 20, pH 8.0) overnight at room temperature, the PVDF
strips were incubated with antiserum against the 42 kDa fusion protein
at 1:500 or with patients' sera at 1:30 for 4 h (1 h at 37°C,
1 h at 4°C, and 2 h at room temperature). Strips were
washed in TBST before addition of the goat anti-rabbit/human
IgG-alkaline phosphatase conjugate (1:40.000) or 2 mCi protein A
125I. In some experiments 10 mM of patients' sera were
preincubated overnight at 4°C with 300 nM of fusion protein before
this last step. Gels were dried and exposed to X-ray film Biomax-MR
(Kodak) at -70°C for 2 days. For phosphatase assay, substrates
5-bromo-4-chloro-3 indoyl phosphate and nitro blue tetrazolium were
added. Molecular weight standards (Bio-Rad prestained) were myosin
heavy chain, 213 kDa; ß-galactosidase, 123 kDa; bovine serum albumin,
85 kDa; ovalbumin, 50.3 kDa; carbonic anhydrase, 33.3 kDa; trypsin
inhibitor, 28.5 kDa; lysozyme, 18.9 kDa; and aprotinin, 7.8 kDa.
Statistical analysis
The effectiveness of sera from different experimental groups in
recognizing the i3 loop in Western blots was compared by using
Fisher's Exact Test. In all cases, differences were considered
significant at P<0.05.
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RESULTS
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A 42 kDa fusion protein comprising the central part
(Arg267 to Arg381) of the
third intracellular loop of the human muscarinic receptor subtype m2
(i3m2AChR) plus the enzyme glutathione S-transferase from S.
japonicum was expressed in E. coli, as described in
Materials and Methods. Despite its abundance in the expression system
used, the fusion protein (GST-i3m2AChR) does not appear to form
insoluble inclusion bodies: it remains soluble when cells are lysed
(Fig. 2A
, lane 1) and can be purified efficiently by
affinity chromatography with yields of 36 µg of protein/ml of the
original culture (Fig. 2A
, lane 3). On Western blots, rabbit
antisera raised against this protein are specific for the m2 subtype
(Fig. 2B
, lane 4); no reaction is observed when m1AChR is
the antigen (Fig. 2B
, lane 3). The presence of antibodies to
the i3-loop fusion protein in the sera of chronic chagasic patients
with progressive myocardiac damage was demonstrated by Western blots
using 125I-protein A. Figure 3
(lanes 3 and 5) shows that the fusion protein is recognized by sera
from chronic chagasic patients representative of groups II (patient
#202) and III (patient #301), but not by sera from patients
representative of group I (patient #105, Fig. 3
, lane 1) and normal
patients (Fig. 3
, lane 7). When patients' sera were preincubated
overnight at 4°C with the fusion protein and then assayed, the
labeled bands were absent (Fig. 3
, lanes 2, 4, and 6), showing the
specificity of the response. The same negative result was observed when
purified GST or m1AChR (Fig. 4
lanes 1 and 3) was exposed to an GST-i3-positive patient serum (patient
#301, group III). If intact m2AChR expressed in CHO cells was used as
the antigen (Fig. 4
. lane 2), a large band at 
87 kDa was
observed, showing that serum from this patient can recognize the whole
glycosylated m2 receptor as well.
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Figure 3. Autoradiography of Western blots of the fusion protein recognized by
sera from chagasic patients. After 15% SDS-PAGE of the
purified fusion protein (10 µg) and its electrotransfer to PVDF
membranes, strips were incubated with the following sera: 1) patient
#105 (group IA); 2) same, preincubated with GST-i3m2; 3) patient #202
(group II); 4) same, preincubated with GST-i3m2; 5) patient #301 (group
III); 6) same, preincubated with GST-i3m2; 7) control serum. All
preincubations were carried out overnight at 4°C.
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Figure 4. Autoradiography of Western blots of GST, m2 and m1 muscarinic receptor
subtypes exposed to serum from a chagasic patient. After 10% SDS-PAGE,
the proteins were electrotransferred to PVDF membranes and strips were
incubated with the serum of patient #301 (group III). The following
proteins were used as antigen: 1) GST (27.5 kDa); 2) 50 µg of m2AChR
(64 87 kDa) expressed in CHO cells; 3) 50 µg of m1AChR (64 70 kDa)
expressed in Sf9 cells in the baculovirus system.
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Table 2
summarizes the results obtained for the sera of all patients. A good
correlation was observed between anti-i3 seropositive response and the
clinical severity of cardiac pathology. Ten percent (1/10) of the
patients in group I, 60% (3/5) of group II, and 80% (8/10) of group
III exhibited a seropositive response. All of the control patients
(6/6) were negative. Patients in groups II and III were not
significantly different, but these two groups together were distinctly
different from controls (P=0.06 and P<0.005).
Patients in group I could not be distinguished from controls on the
basis of this test, but a comparison of group II or group III with
group I showed P values of 0.077 and 0.002 respectively.
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Table 2. Correlation between extent of myocardial damage and seropositive
response to GST-i3m2AChR in chagasic patients and controls
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DISCUSSION
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According to the antigenic mimicry hypothesis, the inflammatory
component of chronic Chagas' disease cardiomyopathy can be explained
by lymphocytes recognizing and producing delayed hypersensitivity
responses toward a tissue-specific heart component bearing structural
similarity to a T. cruzi antigen. Cross-reactive
autoantibodies are directed mainly toward ubiquitous and evolutionarily
conserved molecules, such as myosin, and G-coupled protein receptors
(16
, 17)
. To understand the mechanism underlying the
cardiomyopathy that evolves during the disease, knowledge of the
structure of epitopes recognized by anti-heart specific targets is
required.
The results presented here show that a majority of chronic chagasic
patients in advanced clinical stages of the disease (groups II and III)
have circulating antibodies against the third intracellular portion of
the m2AChR receptor. These antibodies are not detected in most patients
of group I, suggesting that they may contribute to the development of
the abnormalities of cardiac electrogenesis and conduction that are
characteristic of advanced autonomic dysfunction. The specificity of
interaction was ascertained by inhibiting the binding of chronic
chagasic autoantibodies to GST-i3m2 by preincubation with the
recombinant fusion protein GST-i3m2 and by the negative response
obtained when m1AChR subtype or purified GST was used as antigen with
i3 positive serum. The negative response against m1AchR was expected
since the m2 receptor is the only subtype detected in the hearth, the
main impaired tissue in our experiment patients. Furthermore, the third
intracellular loop of m2AChR presents a very low identity (17.4%) when
compared with the corresponding m1AChR region.
That antibodies present in chagasic patients interact with
G-protein-coupled receptors from mammalian cardiomyocytes was first
shown in studies of beat rate and/or force of contraction in isolated
rat atria, first for adrenergic and later for muscarinic receptors
(8
, 9
, 18)
. In the last decade, the second extracellular
loop (o2) of both receptors (a region that is involved in ligand
binding) has been characterized as one of the main immunogenic
epitopes, since there is a strong seropositive antibody response
against the o2 of m2AChRs in asymptomatic chagasic patients with
autonomic dysfunction. In addition, Goin et al. (19)
showed that chagasic affinity-purified anti-o2 IgG from those patients
interacts with muscarinic cholinergic receptor of myocardium to produce
effects on intracellular signal transduction. Among the intracellular
events triggered by these chagasic IgGs are a decrease in atrial
contractility and cAMP formation, functions associated with muscarinic
activity. All of these effects on rat atria by chagasic antipeptide
autoantibodies resemble the effects of the natural neurotransmitter, as
well as those of the total polyclonal IgG, and they are selectively
blunted by atropine as well as being neutralized by synthetic peptides
that correspond in amino acid sequence to o2. The authors suggested
that upon binding to the myocardial neurotransmitter receptors, an
autoantibody behaves like an agonist, producing a persistent stimulus
resulting in muscarinic receptor down-regulation.
The fact that the o2 response is predominantly present in asymptomatic
patients with autonomic dysfunction rather than asymptomatic patients
without alteration of the heart autonomic disorders (both groups with
normal electrocardiograms) led Goin et al. to suggest that these
antibodies could be used as an early marker for heart autonomic
dysfunction.
In contrast, our results show that the i3 antibodies are present in
patients with moderate (group II) to severe (group III) myocardial
damage. In addition, sera from the majority of i3 positive patients
(82%) can reduce cardiac rate and induce AV conduction blockade in
isolated adult rabbit hearts perfused by Langendorff 's method (R.
Pedrosa et al., personal communication).
Taken together, the results with o2 and i3 may mean that a first
autoimmune response triggered by autoantibodies reacting with o2 of the
muscarinic receptors leads to cell damage, which in turn leads to a
polymorphic autoimmune response. As tissue damage increases, i3 is
exposed and a new autoimmune response is triggered.
Autoantibodies against the o2 in the early stages of cardiac damage,
followed by the appearance of autoantibodies against i3 in patients
showing moderate to severe cardiac damage, may lead to a progressive
blockade of myocardial neurotransmitter signal transduction;
autoantibodies against i3 could interfere with intracellular events
such as phosphorylation of the receptor, which is G-protein coupled and
essential for desensitization.
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ACKNOWLEDGMENTS
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We thank Mr. Rui M. Domingues, Miss Rosangela Rosa, and Roberto
Perez Campelo (an undergraduate student) for providing valuable
technical assistance, and Cristina Borges e Sá and Paulo R. Costa
for collecting blood from the chagasic patients. We also thank Drs. M.
Sorenson and Masako O. Masuda for critical reading of the manuscript.
This work was supported by grants from Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq;
PADCT/CNPq; PIBIC), Financiadora de Estudos e Projetos (FINEP),
Fundação José Bonifácio (FUJB), and
Fundação de Amparo a Pesquisa do Rio de Janeiro (FAPERJ).
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FOOTNOTES
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Received for publication December 1, 1998. Revised for publication May 10, 1999.
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ABSTRACT
INTRODUCTION
70 kDa) expressed in Sf9
cells infected with recombinant baculovirus; lane 4: 50 µg of m2AChR
(