retinal dysfunction in patients with chronic chagas

©2006 FASEB

The FASEB Journal express article 10.1096/fj.05-4654fje. Published online January 19, 2006.

Retinal dysfunction in patients with chronic Chagas’ disease is associated to anti-Trypanosoma cruzi antibodies that cross-react with rhodopsin Silvia C. Matsumoto,* Vivian Labovsky,† Marcela Roncoroni,* María C. Guida,† Luisa Giménez,‡ Jorge Mitelman,‡ Horacio Gori,* Renata Jurgelevicius,§ Alejandro Grillo,§ Pablo Manfredi,§ Mariano J. Levin,† and Cristina Paveto†

*Department of Neurology-Hospital Dr. Teodoro Alvarez, Aranguren 2701 (1406), Buenos Aires; †Instituto de Investigaciones en Ingeniería Genética y Biología Molecular UBA-CONICET, Vuelta de Obligado 2490 (1428), Buenos Aires; and ‡Department of Cardiology and §Department of Ophthalmology–Hospital Dr. Teodoro Alvarez, Aranguren 2701 (1406), Buenos Aires, Argentina Corresponding author: C. Paveto, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular UBA-CONICET, Vuelta de Obligado 2490 (1428), Buenos Aires, Argentina. E-mail: [email protected]

ABSTRACT

To investigate retinal involvement in chronic Chagas’ disease, we performed electroretinography and retinal fluorescein angiography studies in chagasic patients. Our results demonstrated a dissociated electrophysiological response characterized by both an abnormal reduction of the electroretinographic b-wave amplitude and a delayed latency, under the dark-adaptated condition. These alterations are compatible with a selective dysfunction of the rods. Antibodies raised against Trypanosoma cruzi that also interact with β1-adrenergic receptor blocked light stimulation of cGMP-phosphodiesterase in bovine rod membranes. The specificity from the antibody-rhodopsin interaction was confirmed by Western blot analysis and antigenic competition experiments. Our results suggest an immunomediated rhodopsin blockade. T. cruzi infection probably induces an autoimmune response against rhodopsin in the chronic phase of Chagas’ disease through a molecular mimicry mechanism similar to that described previously on cardiac human β1-adrenergic and M2-cholinergic receptors, all related to the same subfamily of G-protein-coupled receptors.

Key words: electroretinography ● rod dysfunction

hagas’ disease is a major endemic disease in Central and South America that is caused by the protozoan parasite Trypanosoma cruzi and characterized by an acute and a chronic phase (1, 2).

Based on the results of opthalmological examinations, possible subclinical retinal involvement has been recently suggested in patients with chronic Chagas’ heart disease (cChHD; refs 3, 4).

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Human retina contains the very specialized photoreceptor rod cells, which respond to weak monochromatic light, and cone cells, which respond to colors in white bright light both containing visual pigment rhodopsin (5, 6). Rhodopsin, the apoprotein opsin covalently linked to the light-absorbing pigment 11-cis retinal, belongs to the A family of guanine-nucleotide binding-protein-coupled receptors (GPCRs) and shares common structural, functional, and antigenic properties with the other members of the family (7). The primary photochemical event triggered by light absorption is a rhodopsin conformation change, which starts when a photon is absorbed by the pigment according to its wavelength of maximal absorption, isomerizing from the 11-cis to 11-trans retinal configuration. This activated form of rhodopsin converts the inactive GDP-bound heterotrimeric G protein transducin (Gt-αβγ) to a new active complex with bound GTP, thus dissociating the Gt-βγ subunits. Free Gt-α-GTP activates guanosine 3′,5′-cyclic monophosphate phosphodiesterase (cGMP-PDE). The rapid hydrolysis of cGMP results in the closure of cGMP-gated cation channels in the plasma membrane, thus inducing hyperpolarization and subsequent activation at the cell synaptic terminus (8).

Tissular damage observed in affected organs even in the absence of the intracellular form of the parasite has led most of the researchers to propose an immunological mechanism to explain the pathogenesis of the cChHD (9–12). It has been previously demonstrated that IgG antibodies present in the serum of chronic chagasic patients specifically bind to myocardium β-adrenergic receptor (β-AR) (13) and muscarinic M2 receptor (M2) (14), thus suggesting that the targets of the autoimmune attack include some members of the large GCPRs family. An antigenic mimicry between T. cruzi and some autonomic receptors appears to be responsible for cross-reactivity of antibodies from cChHD patients with normal GPCRs, thus affecting signal transduction and inducing functional and biochemical changes in specific tissues (15, 16). Increasing evidence suggests the potential role of the immune system in the development of optic neuropathies based on the presence of autoantibodies against retinal proteins (17, 18). Along with these findings, we have previously obtained results from opthalmological examinations that strongly suggest that patients with cChHD, frequently present retinal dysfunction (19). In the present work, in order to evaluate the functional and anatomical state of the retina in patients with cChHD, we first performed complete electroretinography (ERG) (20) and retinal fluorescent angiography (RFA) studies (21).

We analyzed the ability of IgG fractions from these patients to recognize rhodopsin. Monoclonal antibodies against bovine rhodopsin (mAb E2) (22), β1-AR (mAb M16) (23), and T. cruzi ribosomal P2β protein (mAb 17.2) (24) were also employed to evaluate the specificity of this interaction. To further investigate the underlying molecular mechanism of the retinal dysfunction evidenced in the electro-ophtalmological studies, we designed a biochemical assay to determine the possible participation of these IgG fractions in the light signal transduction pathway of retinal rod outer segments (ROS). Our findings demonstrate for the first time a selective functional involvement of the rods in patients with cChHD as well as an in vitro molecular interaction between a specific type of IgG fractions from most of these patients and rhodopsin. We present here the results of electrophysiological, biochemical, and immunological investigations together with clinical examinations, as evidence to suggest the relationship between the presence of a specific type of anti-T. cruzi IgG and retinal dysfunction in cChHD patients.


MATERIALS AND METHODS

Patients

Forty-five patients (28 females and 17 males, 26–50 years old) with confirmed cChHD, referred from the Department of Cardiology of the Alvarez Hospital between 1998 and 2002, were selected for evaluations. All of them fulfilled clinical, cardiological, serological, and endemical criteria for the diagnosis of the disease. Every patient underwent complete ophthalmological examinations. The age range selected was 26–50 years old to avoid age-related degenerative changes. Patients with visual acuity under 0.8 in either eye or with acuity differences higher than 0.5 between the eyes were also excluded from the investigation. Fifty healthy individuals, 27 females and 23 males aged from 25 to 55 years old, were included as control patients.

Samples

The IgG fractions from sera from both healthy controls and cChHD patients were prepared as described in previous reports (24). Briefly, the IgG fractions of the sera were prepared by a dilution of 1/5 of the serum in PBS and precipitation at 40% ammonium sulfate saturation. The precipitate was redissolved in PBS and dialyzed exhaustively against PBS at 4°C. Protein concentration was quantified by Bradford reagent method (25), and the immunoreactivity was typified by ELISA (26). IgG fractions functional activity was evaluated as either β1-AR or M2-ChR cross reactive by using the cultured neonatal heart myocytes test system as previously reported (26).

Electrophysiological studies

Electrophysiological recordings were performed with an ATI Nautilus with conventional techniques following standard criteria for clinical electroretinography determined by the ISCEV. The temporal properties of the ERG response are usually defined by the time-to-peak of the b-wave and are measured from stimulus onset to the peak of the b-wave (27). Full-filled Ganzfeld monocular stimulations were performed and electroretinographic responses were recorded by an active contact electrode located over the cornea. Skin contact electrodes were located near each orbital rim as reference and in the middle forehead as ground electrode, with impedances <5 K Ohms in all of them. The studies were designed to obtain separate rod and cone responses for each examined eye. Rod responses were recorded after 15 min of dark adaptation, which in all cases lead to maximal natural and symmetrical pupil dilatation. Monocular stimulation was performed successively with a dim red, dim blue, and white flashes at a frequency of 0.1 Hz, low enough to avoid excessive adaptation and with an interval of 5 min rest among the different trials. Cone responses were recorded in the same way but after 10 min of light adaptation with a background luminance of 10 fL. The analysis time was 100 msec, filters were set from 1000 to 10 Hz, and 25 responses were averaged at least twice for each trial. The obtained a-wave, b-wave latencies, and b-wave amplitudes were measured and the values were compared statistically (Student’s t test) to those of 50 healthy controls (27 females and 23 males, 25–55 years old). Electrophysiological studies were complemented with complete standard ophthalmological evaluation and RFA studies.


Preparation of parasite lysates

Samples of T. cruzi trypomastigotes, Tul 2 strain, obtained from blood of infected mice, were lysed by osmotic shock and sonication and resuspended in PBS at 2 × 106 parasites/ml (26).

Peptides

Peptides were synthesized as described (26, 28). R13 peptide corresponds to the main epitope of the T. cruzi P2 β ribosomal protein, mapped to the C-terminal aminoacid sequence (TcP2β, 107–119, EEEDDDMGFGLFD); H26R peptide corresponds to the sequence of the second extracellular loop of the human β1-AR (Hβ1-AR, 197–222, HWWRAESDEARRCYNDPKCCDFVTNR); and E2p is a peptide derived from the second extracellular loop of the bovine rhodopsin (bovine rhodopsin, 182–199, GMQCSCGIDYYTPHEETN).

Production and characterization of monoclonal antibodies

The monoclonal antibodies M16 raised against H26R peptide and 17.2 against R13 were obtained according to standard procedures (23, 24, 29). Splenocytes of Balb/C mice immunized with H26R or recombinant TcP2β were fused with the SP2/0 myeloma cell line in a ratio of 1:10 by using ClonaCell-HY kit (StemCell Technologies, Vancouver, BC, Canada). Hybridoma cloned were tested for production of antibodies against the respective immunogenic peptide by ELISA. Positive clones for H26R peptide were cultured in Iscove’s medium supplemented with 10% fetal calf serum (FCS; Bio Wittacker Europe, Verviers, Belgium), 2 mM glutathione, 1 mM sodium pyruvate, 100 UI/ml penicilline-streptomycine, and 50 μM β-mercaptoethanol. Purification of immunoglobulins was carried out by affinity purification with an affinity column made up by coupling the peptide to the CNBr-activated Sepharose (Pharmacia, Uppsala, Sweden) by standard procedure (23). On the other hand, clones producing antibodies against R13 peptide were amplified in ascite fluids by intraperitoneal injection into histocompatible mice previously primed with 0.5 ml pristane (Sigma) (24). Ascite fluid was collected, and immunoglobulins were precipitated with 40% (NH4)2SO4 saturation. Protein concentration was evaluated by measuring the absorbance at 280 nm (1.40 for a 1 mg/ml solution). The fine specificity was assessed according to described previously epitope mapping using biotinylated R13 peptide. The agonist-like monoclonal antibody against the M2 acetylcholine receptor was raised against a peptide corresponding to the second extracellular loop of the receptor as reported by Elies et al. (30). Monoclonal antibody E2 was kindly given by Dr. Karren Hyde upon request to Dr. Heidi Hamm (Vanderbilt University, Nashville, TN; refs 22, 31).

Preparation of bovine rod outer segment membranes

Fresh bovine eyes were employed as source of rods. ROS disk membranes were prepared according to Papermaster and Dreyer (32) with slight modifications. All manipulations were performed under dim red light illumination. The dark-adapted bovine retinas were removed and carefully isolated from the pigmented epithelium. The retinas were collected in Sorvall SS34 centrifugation tubes and homogenized by vortexing for 1 min in 10 ml of 5 mM-Tris HCl pH 7.4, 65 mM NaCl, 2 mM MgCl2, and 40% sucrose (buffer A). After centrifugation at 4000 g for 4 min, the ROS-containing supernatant was diluted in 20 ml of 50 mM Tris-HCl pH 7.4, 1 mM MgCl2 (buffer B) and centrifuged at 14,000 g for 40 min. Pellet was resuspended in buffer B,


supplemented with 0.77 M sucrose and disrupted by one passage through a 20-G (∼0.9 mm) needle fitted to a syringe. The suspension was layered on the top of a step gradient of 0.84 M, 1.00 M, and 1.14 M sucrose in buffer B and centrifuged at 27,000 g for 70 min. ROS membranes were obtained from the interfase 0.84 M/1.00 M, pelleted by centrifugation at 17,000 g for 30 min; washed twice with buffer B and resuspended in 20 mM Tris-HCl pH 7.6, 50 U/ml aprotinine, 100 μg/ml leupeptin, 10 mM pepstatin, and 0.5 mM phenylmethylsulfonylfluoride (PMSF), aliquoted, and stored at −70°C. Rhodopsin concentration was determined spectrophotometrically (33) based on the molar extinction coefficient value (ε498) of 40,000 M−1·cm−1.

Phosphodiesterase activity assay

Phosphodiesterase activity was measured according to Farber and Lolley (34) with modifications. The hydrolyzed [3H]cGMP was quantified as described previously (35). cGMP-PDE activity was measured under darkness for 5 min (basal), and light-induced activity was assayed in samples illuminated with steady white light for 5 min (48.6 μmol photon/m2/sec) under a stable incubation temperature of 30°C in all the samples; 0.15 μg of ROS membrane preparation were used throughout. Controls assays were performed to assess that no significant shift from 500 to 380 nm was produced on the absorbance peak of rhodopsin after incubation of ROS membranes with IgG fractions or mAbs under darkness at 4°C. This preincubation had no effect on cGMP-PDE basal activity. Results were expressed as enzyme units per milligram of total protein. One unit of phosphodiesterase activity is defined as the amount of enzyme that catalyzes the hydrolysis of 1 nmol of cGMP/min.

SDS-PAGE analysis and immunoblot analysis

Twenty micrograms of ROS membrane per well were solved in 12% SDS-PAGE (36) electrophoretically transferred to a nitrocellulose membrane. Immunodetection of rhodopsin was performed with 150 nM of mAb E2, of mAb M16 or of mAb 17.2 for 1 h at 4°C. To evaluate cross reaction of IgG fractions from chagasic patients with rhodopsin, 100 μg/ml of IgG for 1 h at 4°C was used. The interaction between both control and IgG fractions from cChHD patients and blotted rhodopsin was competed with either 1 mg/ml of T. cruzi lysate or 100 μM each peptide. Bound antibodies were detected by using horseradish peroxidase-labeled goat anti-mouse IgG (H+L) diluted 1/3000 (v/v) (Sigma-Aldrich) for 1 h at 4°C and revealed with 3,3′,5,5′–tetramethylbenzidine dihydrochloride preparation (TMB; Sigma-Aldrich).

RESULTS

Clinical evaluation of patients with cChHD

We performed a thorough clinical and cardiological evaluation of 45 cChHD patients (Table 1). None of them suffered peripheral isquemic dysfunction and/or neurological complications that could influence the outcome of the ERG studies. Only 15 out of the 45 selected patients (33.33%) spontaneously referred diminished visual acuity under dim light, a symptom that was not explained by conventional ophthalmological evaluation.


Electrophysiological studies

We employed ERG and RFA to evaluate the functional and anatomical state of the retina in the selected cChHD patients and healthy individuals. The responses of patients evoked by all three white, red, or blue flash stimulations under the light-adapted state were similar to, or slightly weaker than, the normal-shaped and high amplitude responses of the controls. However, under dark-adapted conditions using either dim blue or dim red flash stimulations, we observed a temporal dispersion of each response with a remarkable reduction of the b-wave amplitude in cChHD patients when compared with controls (Fig. 1; Table 2). Dim blue flash stimulation presented weak, delayed, or even nonexistent responses in the b-wave amplitude, at least in one eye of 36 out of 45 patients (80%). This failure was bilateral only in 9 out of 45 patients (20%; Fig. 1; Table 2). Dim red flash stimulation evoked abnormal responses for b-wave latency and b-wave amplitude in 37 out of 45 patients (82.22%) in one eye and 19 of the 45 patients (42.22%) in both eyes (Fig. 1; Table 2). These abnormal responses tended to improve when dark adaptation time was prolonged up to 45 min, suggesting a time-dependent adaptation rather than an all-none response type deficit. In the present studies, the blue and red flashes were dim enough for the a-wave amplitude not to be discerned leaving only the rod-dominated slower b-wave. In accordance, the a-wave appeared included in the b-wave amplitude (37). Retinal fluorescent angiography was performed in 33 of the patients with abnormal ERG. Parafoveolar retinal pigment epithelium dispersion, general pallor, and atrophy were observed in the retinal epithelium of all these patients. In 28 out of 33, these abnormalities were more evident at the posterior pole (Fig. 2).

IgG fractions from cChHD patients with confirmed retinal dysfunction target visual pigment rhodopsin

To elucidate the molecular mechanism underlying the abnormal ERG responses registered in the studies described above, we first examined by Western blot the possible interaction between IgG fractions from cChHD patients with retinal dysfunction and rhodopsin from bovine ROS disk membranes. IgG fractions from these patients recognized the 40 kDa protein band corresponding to bovine rhodopsin (Fig. 3A), while IgG fractions from normal control did not reveal any band in the blotted membranes. We identified the 40 kDa protein band as rhodopsin by immunoblotting with the mAb E2 (Fig. 3A and B). This reaction was abolished by preincubation of mAb E2 in the presence of the E2 peptide (E2p) (Fig. 3B). We also probed immunoblots of ROS preparations with the mAb M16 directed against the second extracellular loop of the human β1-AR (21). This antibody recognized rhodopsin (Fig. 3B). The same result was obtained with the mAb 17.2, an antibody that reacts with the C-terminal epitope of the T. cruzi ribosomal P2β protein, peptide R13 (22; Fig. 3B). We confirmed the specificity of each anti-rhodopsin reaction by preincubation of mAb M16 and 17.2 with peptides H26R and R13, respectively (Fig. 3B). Experiments in Fig. 3B show that mAb raised against β1-AR cross react with rhodopsin. Accordingly, to assess a presumed correlation between β1-AR immunoreactivity of IgG fractions from cChHD patients and the ability to recognize rhodopsin, we probed ROS immunoblots with IgG fractions from cChHD patients that also stimulated the β1-AR in the neonatal cardiomyocytes assay (26). IgG fractions with an exclusive anti-β1-AR reactivity strongly recognized rhodopsin (Fig. 3C). On the contrary, IgG fractions previously shown to have selective anti-M2 reactivity did not (26; Fig. 3C).


Thereafter, IgG fractions from cChHD patients with selective retinal dysfunction were preincubated with T. cruzi homogenate, which strongly diminished rhodopsin detection (Fig. 3C). We could also observe the competitive effect of R13 peptide (28), as well as that of E2p peptide, on the recognition of rhodopsin by these IgG fractions (Fig. 3C).

Participation of IgG fractions from cChHD patients in bovine retina light signal transduction: Functional assay of rhodopsin

We developed an assay to determine whether IgG fractions from cChHD patients could affect rhodopsin-mediated light signal transduction pathway in bovine ROS membranes (38). This assay consists of the determination of cGMP-PDE activity under different conditions: basal activity, light stimulated activity and in presence or absence of anti-T. cruzi antibodies. Light stimulation of cGMP-PDE in this system is referred to as the difference between the enzymatic activity measured under white light illumination and the enzymatic activity measured under darkness (basal activity) (Fig. 4A and B, inset). Basal activity of cGMP-PDE was not modified by incubation with IgG fractions or any mAb under darkness for 30 min at 4°C. The mAb E2 inhibited light stimulation of cGMP-PDE activity (Fig. 4A and B). The inhibition was strongly reverted by preincubation with E2p peptide (Fig. 4A). IgG fractions from cChHD patients that presented an exclusive β1-AR cross-reactivity blocked rhodopsin-mediated light stimulation of cGMP-PDE activity (Fig. 4A, 8–15), whereas IgG fractions with a clear anti-M2 cross-reactivity exerted no measurable effect (Fig. 4A, 6–7). IgG fractions from healthy control individuals did not inhibit the enzymatic activity (Fig. 4A, 1–5). The inhibitory effect of IgG fractions cross-reactive to β1-AR from cChHD patients was abolished by preincubation with either T. cruzi lysate (Fig. 4A, 16–23) or peptides R13 (Fig. 4A, 24–25) and H26R (Fig. 4A, 26–27). The T .cruzi lysate had no influence on the stimulation of the enzymatic activity (Fig. 4A, 28). The light-stimulated cGMP-PDE activity was also strongly inhibited by mAbs M16 and 17.2 (Fig. 4B). However, antibodies specifically reacting with M2 receptor did not produce the same inhibition (Fig. 4B). The inhibitory effect of the mAbs M16 and 17.2 was antagonized by the presence of H26R and R13 peptides, respectively (Fig. 4B).

Correlation between electrophysiological abnormalities and light signal transduction interference

Finally, in order to correlate the altered ERG response with the molecular findings presented above, we tested the ability of the IgG fractions from cChHD patients that presented retinal dysfunction to interfere in light stimulation of rhodopsin-dependent cGMP-PDE from bovine ROS membranes (Table 3). We found that all these IgG fractions blocked with different intensities light stimulation of the enzyme. IgG fractions from cChHD patients with normal ERG response failed to inhibit cGMP-PDE stimulation, thus showing the same behavior as the IgG fractions from healthy individuals. Notably, IgG fractions positive for rhodopsin in both Western blot and functional assays, also had β1-AR-stimulating activities as assessed on cultured neonatal cardiomyocytes. IgG fractions from cChHD patients with normal ERG were either M2 cross reactive or did not cross react at all with either β1-AR or M2 receptors, as assessed by ELISA and the neonatal cardiomyocytes functional assay.


DISCUSSION

ERG evaluation of patients with cChHD demonstrated a dissociated clinical retinal involvement with selective dysfunction of the rods in eighty percent of these patients (Table 2). Abnormalities in the parafoveolar peripheral retinal pigment epithelium that correlate with the anatomical distribution of the rods (39) were confirmed in ~73% of the patients with anomalous ERG (Fig. 2). Interestingly, IgG fractions from cChHD patients with retinal dysfunction reacted with blotted rhodopsin (Fig. 3A). The inhibition of light-induced cGMP-dependent PDE activity by IgG fractions from these patients suggests their functional interaction with rhodopsin. The attenuation of this functional inhibition by T. cruzi lysates and both the parasite R13 peptide and the β1 adrenoceptor H26R peptide, accounted for the specificity of this antibody-mediated photoreceptor blockade. On average, the magnitude of interference on light signaling (Fig. 4A; Table 3) by IgG fractions from cChHD patients correlate with the magnitude of abnormalities of electro-ophthalmological records (Fig. 1; Table 2). The fact that these IgG fractions also exerted a β1 adrenergic stimulating effect on neonatal cardiomyocytes gives additional support to our results.

Detailed molecular analysis of this type of reactivity has previously demonstrated that IgG from cChHD patients recognize a sequence on the C-terminal region of the T. cruzi ribosomal P proteins, namely R13, which shares homology with an epitope on the second extracellular loop of the human β1-AR, AESDE peptide (40). Notably, the recognition of β1-AR by human anti-R13 antibodies is associated to their ability to interact with non contiguous acidic residues of the R13 epitope (26). This is also the case of mAb 17.2, which reacts with the C-terminal of TcP2β protein (24). On the contrary, Mahler et al. (26) demonstrated by epitope mapping that M2 stimulating antibodies showed higher affinity for a continuous disposition of acidic residues. Rhodopsin shares structural, functional, and antigenic properties with GPCRs, including adrenergic and cholinergic receptors, all of them transducing extracellular signals through interactions with G-proteins (41). Human and bovine rhodopsin shares 93.4% of sequence homology with partial conservation of aminoacidic residues at the second extracellular loop and the site of attachment for 11-cys-retinal (e.g., Glu-181, Asp-190, Glu-197, and Glu-201; ref 42). The conserved acidic residue Glu-181 could play a crucial role in the rhodopsin family in shifting the absortium maximun, as demonstrated by Terakita et al. (43). These data support our experimental results where only IgG fractions against T. cruzi with β1-AR affinity inhibit signal transduction in rods. Monoclonal antibodies M16 and 17.2 interact with different epitopes, as demonstrated by epitope mapping in Mobini et al. (23) and in Mahler et al. (26) respectively, producing the same functional response.

The inhibitory effect of light activation of rhodopsin-dependent cGMP-PDE, by the interaction of mAb M16, mAb 17.2, and mAb E2 with rhodopsin, may also be explained by the recognition of different epitopes in the second extracellular loop of rhodopsin that might result in the same functional effect. Although we cannot demonstrate the precise residues that constitute the epitopes for the inhibitory interaction of IgG fractions from cChHD patients, we propose that the results in Figs. 3 and 4 may be responses to the recognition of different epitopes, resulting in the same ability to block light signal transduction. The result of our functional assays showed an antagonist effect of IgG fractions from cChHD patients on light stimulated cGMP-PDE activity and not a partial agonist effect. Otherwise, Ghanouni et al. (44) has already described that the energy necessary to activate rhodopsin comes exclusively from light. Moreover, in contrast to


the large number of receptors that are activated by the binding of diffusible agonist ligands, rhodopsin is activated by generation of the agonist all-trans-retinal from 11-cis-retinal through photoisomerization into the opsin moiety (45).

There is growing evidence of a possible link between autoantibodies against retinal proteins and ophthalmological dysfunction. High levels of anti-rhodopsin antibodies in patients with normal pressure glaucoma (NPG) as well as sequence similarities between rhodopsin and numerous proteins from pathogenic bacteria and virus have been demonstrated (17, 18). Their findings give additional evidence for molecular mimicry and generation of pathogenic anti-rhodopsin antibodies in opthalmopathies. Although sequences of β1-AR and rhodopsin are not identical, it is worthy to point out that the cross-reactivity between antibodies and antigens depends not only on the conservation of critical residues in the precise combining site but also on the influence of ionic interactions (46). The recently solved crystalline structure of rhodopsin has confirmed that the binding site of the chromophore lies within a functional region named chromophore binding pocket (47). The pocket-neighboring regions may become targets of anti-T. cruzi antibodies from cChHD patients. Analysis of the localization of the second extracellular (EC-II) loop of bovine rhodopsin shows that it is closed over the transmembrane regions by a disulfide linkage between Cys-110 and Cys-187 (48), allowing exposure of the acidic residues Glu-196 and Glu-197 (in human rhodopsin Glu-197). Since loop E2 folds back into the membrane-embedded domain of the receptor to form part of the binding pocket for the 11-cis-retinylidene chromophore, an antibody that is able to recognize exposed acidic residues may influence directly the absorption properties of the visual pigment. Antibodies that bind to these sites involved in conformational changes block receptor activation by disturbing the chromophore-protein interaction (49) and consequently could eventually interfere in signal transduction in rods. Parasites as T. cruzi appear to be able to induce a pathological autoimmune response against the photopigment rhodopsin of the rods in a similar molecular mimicry mechanism to that described on cardiac human β1-AR (50, 51). Moreover, it has been demonstrated that the anti-T. cruzi antibody levels with a marked β-stimulating activity correlate with the degree of clinical involvement (52).

Our results suggest that both IgG fractions from cChHD patients and mAbs M16 and 17.2 would also recognize retinal rhodopsin, inhibiting rhodopsin-mediated signal transduction of bovine rods (Fig. 3A and B). A correlation between alterations of both b-wave latency and b-wave amplitude parameters of ERG, abnormal RFA, and the presence of antibodies specifically reactive to rhodopsin in cChHD patients sera could then be suggested. The results presented here indicate that T. cruzi is able to induce a pathological response not only against cardiac human β1-adrenergic and muscarinic M2 receptors but also against the photoreceptor rhodopsin. Here we propose a model that could eventually explain the inhibition of light signal transduction by antibodies against the parasite present in the sera of cChHD patients with retinal dysfunction (Fig. 5).

ACKNOWLEDGMENTS

We specially wish to thank Dr. Mirtha Flawia and Dr. Hector N. Torres for their scientific support. We are grateful to Heidi Hamm and Karen Hyde for kindly providing the mAb E2. We also express our gratitude to Dr. Maria L. Cantore for interest and helpful criticism of the manuscript. This work has been supported by grants from the World Health Organization/Special Program for Research and Training in Tropical Diseases, Fundacion Bunge y Born (Buenos


Aires, Argentina), and the National Research Council (CONICET, Buenos Aires, Argentina). The work of M. J. Levin is partially supported by an International Research Grant of the Howard Hughes Medical Institute (Chevy Chase, MD).

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Received July 20, 2005; accepted November 7, 2005.


Table 1 Clinical findings in chagasic patients

Sex 28 F 17 M

Age (yr) 38 ± 12.5 42 ± 8.8

Cardiovascular findings

Hypertension (mild) 12 13 Sinus bradycardia 0 2 First degree A-V block 2 1 Atrial extrasystolia 2 1 Right bundle branch block (RBBB) 20 5 RBBB + left anterior bundle hemi-block 8 12 Isolated ventricular extrasystolia 2 4 Gastroinstestinal findings

Mega esophagus 1 0 Mega colon 2 0

Others

Hyperthyroidism 4 0

Clinical and cardiological evaluation was performed to discard any systemic pathology that could lead to alteration of electroretinographic (ERG) studies F: female; M: male.


Table 2 Registered data of a-wave and b-wave latencies and peak to peak b-wave amplitude between control and patients with chronic Chagas’ heart disease

ERG Response

Stimulus

Patients

a-wave latency (ms)

b-wave latency (ms)

b-wave amplitude (microvolts)

recording ratio

Light-adapted condition-White

flash

NORMAL (n=100 eyes)

cChHD (n=90 eyes)

17.33 ± 0.95 18.20 ± 2.18

38.10 ± 2.20 39.34 ± 2.46

17.12 ± 6.50 15.88 ± 6.60

100/100 90/90

Dark-adapted condition-Dim blue

flash

NORMAL (n=100 eyes)

cChHD (n=90 eyes)

17.75 ± 2.00 19.44 ± 3.80

38.22 ± 2.88 43.18 ± 4.48

15.50 ± 5.35 *6.15 ± 3.88

100/100 63/90

Dark-adapted condition-Dim red

flash

NORMAL (n=100 eyes)

cChHD (n=90 eyes)

18.44 ± 2.48 20.82 ± 3.12

17.49 ± 3.20 *44.12 ± 5.18

14.45 ± 4.45 *6.26 ± 3.12

100/100 55/90

Values are mean of data obtained from the recorded eyes ± SD. *P < 0.001.


Table 3 IgG fractions from cChHD patients with rod retinal dysfunction inhibited light stimulation of cGMP-PDE activity in ROS bovine membranes

IgG Fractions Rhodopsin-Dependent cGMP-PDE Units

Not added 92 ± 9.34

Normal patients 1−10* 87 ± 7.65

1 79 ± 5.31

2 81 ± 9.87

3 89 ± 8.40

4 75 ± 6.81

cChHD patients with normal ERG response

5 87 ± 4.32

6 33 ± 1.20

7 41 ± 8.01

8 36 ± 6.51

9 28 ± 3.23

10 22 ± 3.68

11 23 ± 9.80

12 38 ± 3.21

13 17 ± 1.12

14 45 ± 2.32

cChHD patients with abnormal ERG response

15 28 ± 4.35

*Result is average of 10 IgGs from normal patients evaluated. Values are mean ± SD of at least 3 independent determinations.


Fig. 1

Figure 1. Comparison of recorded ERGs from normal and chronic Chagasic patients. Normal ERG evoked response is shown above; b-wave amplitude evoked by dim blue and red flashes under dark-adapted state strongly diminished in patients with chronic Chagas’ heart disease, but b-wave latency showed temporal dispersion only when stimulated with dim red flash (Table 2).


Fig. 2

Figure 2. Retinal fluorescein angiography of patients with chronic Chagas’ heart disease. Left panel) Retinal pigment epithelium of the eye of a normal healthy patient. Right panel) Abnormal retinal pigment epithelium dispersion and mild scattered defects associated to a general pallor more marked at its posterior pole observed in the eye of a cChHD patient.


Fig. 3

Figure 3. Immunorecognition of rhodopsin in ROS membrane preparation. A) IgG fractions from cChHD patients that presented abnormal ERG revealed bands showing a band of 40 kDa, the expected mobility for bovine rhodopsin as indicated by the specific mAb E2. B) Monoclonal antibodies M16 and 17.2 recognized rhodopsin in blotted membranes. The band corresponding to rhodopsin could not be revealed after specific competition of mAbs E2, M16, and 17.2 with E2, H26R, and R13 peptides. Monoclonal antibody against M2 did not recognize rhodopsin. C) IgG fractions cross-reactive to β1-AR from cChHD patients strongly reacted with rhodopsin, while IgG fractions with M2 affinity from cChHD patients did not; the specificity of immunorecognition was demonstrated by simultaneous incubation of IgG fractions from cChHD patients with retinal dysfunction and T.cruzi lysate (Tc), R13 peptide, or E2 peptide.


Fig. 4

Figure 4. Monoclonal antibodies and IgG fractions from cChHD patients inhibit light induction of rhodopsin-dependent cGMP-PDE enzymatic activity from bovine ROS membranes. Enzymatic activity was performed on 1.5 µg/ml of ROS membranes preparation (0.5 µM rhodopsin) in a final volume of 100 µl. A) IgG fractions from control patients scarcely inhibited cGMP-PDE activity (lanes 1–5). No inhibition of rhodopsin dependent cGMP-PDE activity was produced by IgG reactive to M2 (lanes 6–7). IgG fractions from cChHD patients reactive to β1-AR significantly reduced the enzymatic activity (lanes 8–15), which was restored when preincubated with T.cruzi total antigen (lanes16–23). Reversion of inhibitory effect is also demonstrated with R13 (lanes 24, 25) and H26R (lanes 26, 27) peptides. No effect on enzyme activity light stimulation is produced by T.cruzi lysate (lane 28). B) Light-induced cGMP-PDE activity is strongly inhibited when preincubated with mAb E2. Magnitude of inhibition caused by mAb M16 was similar to that produced by the specific mAb E2. This inhibition was abolished when mAb E2 and M16 were preincubated with E2 and H26R peptides. Monoclonal antibody 17.2 showed similar inhibitor effect, which diminished by preincubation with R13 peptide. No inhibition was observed with mAb M2. Inset, rhodopsin-dependent cGMP-PDE activity is expressed as difference between light-stimulated activity (■) and basal activity (□) recorded under continue slow intensity red light (dark condition). Results shown here are mean of at least 3 experiments. SD was calculated with Graph Pad Prism software.


Fig. 5

Figure 5. Hypothetic model to illustrate the molecular events underlying retinal pathology of cChHD patients. T. cruzi induces a pathological autoimmune response against the photopigment rhodopsin through a molecular mimicry mechanism related to common antigenic, structural and functional properties between the members of same subfamily of GPCRs. Some of these antibodies could specifically interfere with critical structures of rhodopsin and block light-induced signal transduction. Resulting inhibition of functional activation of the rods would be compatible with clinical and electrophysiological findings observed in cChHD patients.


retinal dysfunction in patients with chronic chagas

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