Cruzipain induces autoantibodies against cardiac muscarinic acetylcholine receptors. Functional and pathological implications

2.1 Detection of antibodies against myocardial mAChR in sera from cruzipain-immunized mice

Cruzipain-reactive antibodies that are also reactive to heart antigens have been reported 16. To test the ability of anti-cruzipain IgG to interact with myocardial mAChR, a competition-binding assay was performed on normal mouse cardiac membranes. Fig. 1 shows that anti-cruzipain IgG inhibited the specific mAChR antagonist, [³H]-QNB (quinuclidinyl benzilate), binding in a concentration-dependent manner, whereas normal IgG did not. Saturation studies and computer-assisted fitting curves showed that 1×10^–7 M anti-cruzipain IgG interacted irreversibly, decreasing the number of binding sites (B_max) without changes in the equilibrium dissociation constant (K_d). In contrast, control IgG affected neither B_max nor K_d (Table 1).

In previous work, it has been shown that IgG from chagasic patients selectively interacts with the second extracellular loop of human M₂ mAChR. Therefore, to evaluate the molecular interaction between anti-cruzipain IgG and this receptor, the 24-mer synthetic peptide, M₂e2 (corresponding to the second extracellular loop), was used as coating antigen in ELISA. Fig. 2A shows an increase in optical density (O.D.) values in a concentration-dependent manner using anti-cruzipain sera. Values obtained with immune sera were always >3 standard deviations above those of control IgG. Reaction specificity was assessed by the ability of the anti-M₂e2 IgG purified from the corresponding sera to increase the O.D. values (Fig. 2B). Affinity-purified anti-M₂e2 IgG (7×10^–8 M) maximally increased O.D. values; this concentration had an equivalent effect to 5×10^–7 M of total anti-cruzipain IgG. The excluded fraction (i.e. the fraction excluded from M₂e2 affinity-columns) showed O.D. values similar to control IgG. Incubating the antibodies with the synthetic peptide, M₂e2, neutralized the specific reaction of the affinity-purified anti-M₂e2 IgG derived from the anti-cruzipain serum.

2.2 mAChR-mediated effect of anti-cruzipain IgG

To evaluate the effect of anti-cruzipain IgG on myocardial intracellular signals coupled to M₂ mAChR, changes in cAMP levels, inositol phosphate (IP) accumulation, nitric oxide synthase (NOS) activity and cGMP production were measured.

Mice atria were exposed to 1×10^–7M anti-cruzipain IgG for different times to accurately determine its kinetic behaviour and the time for their maximal effect. As shown in Table 2, there was a significant decrease in atrial cAMP exposed to anti-cruzipain IgG or the corresponding affinity-purified anti-M₂e2 IgG. The maximal decrement was obtained at 2–3 min and persisted for 10 min (Fig. 3). Furthermore, it was blocked by AF-DX 116 (1×10^–6 M). Effective neutralization was also observed when preincubating the anti-cruzipain IgG with M₂e2. The effect of anti-cruzipain IgG and anti-M₂e2 IgG resembled that of the authentic agonist, carbachol, at 5×10^–8 M. Control IgG or the M₂e2-excluded IgG fraction had no effect on intracellular cAMP levels.

To assess the action of anti-cruzipain IgG on phospholipase C (PLC)-induced intracellular second-messenger production in cardiac preparations, atria were incubated with [myo-³H]-inositol ([³H]MI). Thereafter, water-soluble radiolabeled IP levels formed in the presence or absence of anti-cruzipain IgG or affinity-purified anti-M₂e2 were determined. Table 3 shows that total anti-cruzipain IgG and the corresponding anti-M₂e2 IgG were able to produce a significant increase in phosphoinositide formation. The effect of the immune IgG was similar to carbachol action. The anti-M₂e2 IgG effect could be abolished by pre-treating cardiac tissue with AF-DX 116 or U-73122, indicating that M₂ mAChR and PLC-mediated hydrolysis of IP were involved in this effect. M₂e2 also neutralized the IgG action, indicating the specificity of the reaction (Table 3).

Knowing that the endogenous nitric oxide signaling system is involved in activation of atrial M₂ mAChR 18, we analyzed whether the immune IgG led to increased NOS activity and production of cGMP on murine myocardium. As can be seen in Fig. 4, the affinity-purified anti-M₂e2 increased both NOS activity and cGMP production in a concentration-dependent manner, whereas the excluded IgG fraction (i.e. the fraction with no affinity for M₂e2) showed no effect.

To verify the nature of the mechanism involved in increased cGMP synthesis and NOS activity, mice atria were incubated with several inhibitors. Table 4 shows that AF-DX 116 inhibited the stimulatory action of the affinity-purified anti-M₂e2 IgG on both cGMP production and NOS activity. Furthermore, inhibition of PLC (by U-73122), calcium calmodulin (by TFP) and NOS (by N^G-monomethyl-L-arginine [L-NMMA]) blunted M₂ mAChR-dependent activation of NOS activity and cGMP accumulation. Additionally, the natural substrate L-arginine completely reversed the inhibition of L-NMMA. As control, the increment of cGMP was abolished by the inhibitor of soluble guanylate cyclase (ODQ). Moreover, carbachol (5×10^–8 M) and anti-cruzipain IgG mimicked the effect of the specific anti-M₂e2 IgG. Control IgG had no effect.

2.3 Functional effects of anti-cruzipain antibody

As seen in Figs. 5 and 6, addition of anti-cruzipain IgG and specific anti-M₂e2 IgG results in a decrease of atrial contractility and frequency, the specific affinity-purified anti-M₂e2 IgG being more potent than anti-cruzipain total IgG. Control IgG showed no effect in the studied system (Figs. 5 and 6, upper panels).

The functional effects of anti-cruzipain IgG were neutralized by the same factors (AF-DX116 and M₂e2) that inhibited signal transduction coupled to M₂ mAChR (Figs. 5 and 6, lower panels).

To determine if an endogenous nitric oxide signaling system participates in the negative inotropic action of anti-cruzipain IgG, isolated mouse atria were incubated with inhibitors of NOS activity and cGMP synthesis. Fig. 5, lower panel, shows that L-NMMA inhibition of NOS activity or inhibition of soluble guanylate cyclase by ODQ prevented the inotropic negative effect of anti-cruzipain IgG. On the other hand, db-cAMP (a cAMP analogue) was able to reverse the negative chronotropic effect of the anti-cruzipain IgG (Fig. 6, lower panel).

These results indicate that the negative inotropic effect is related to the NO-cGMP pathway, whereas the negative chronotropic action is related to intracellular cAMP reduction triggered by anti-cruzipain antibodies.

4.1 Cruzipain purification

Epimastigote forms of T. cruzi Tulahuen strain were grown at 28°C in brain heart infusion medium (Becton Dickinson) supplemented with 0.5% tryptose, 10% FCS, 200 mg/ml hemin and 100 U/ml streptomycin. Parasites were harvested at the exponential growth phase, centrifuged at 5,000×g for 10 min at 4°C, and washed with PBS. Parasites were resuspended in three volumes of 0.25 M sucrose and 5 mM KCl; next the irreversible protease inhibitors, 1 mM N-α-p-tosyl-L-lysine chloro methyl ketone and 1 mM PMSF (phenylmethylsulfonylfluoride) (Sigma) were added. Epimastigotes were disrupted by three cycles of freezing (–20°C) and thawing (4°C). The homogenates were centrifuged at 7,000×g for 15 min at 4°C. Saturated ammonium sulfate solution, adjusted to pH 7.1 with NH₄OH, was added at 50% saturation to the supernatant, with stirring in an ice bath. The precipitate obtained after centrifugation of this suspension was carefully dissolved and dialyzed in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4. CaCl₂, MgCl₂ and MnCl₂ were added to a final concentration of 5 mM each. Subsequently, the samples were submitted to affinity chromatography. The absence of enzyme activity was checked by 10% SDS-PAGE containing 0.1% gelatin as substrate. It was important to eliminate cruzipain activity to avoid the inflammatory properties of cruzipain as a proteinase 4. After electrophoresis performed at 120 V, the gel was incubated with buffer — 50 mM sodium phosphate, pH 5.7, at 37°C overnight — and it was then stained with Coomassie brilliant blue R250. Samples were neither reduced nor boiled.

4.2 Immunization of BALB/c mice

Inbred female BALB/c mice, aged 6–8 weeks (purchased from Centro Nacional de Energía Atómica, Buenos Aires, Argentina), were immunized by intradermal injection, at the base of the tail and along the back, with cruzipain that had been irreversibly inactivated and was emulsified in complete Freund's adjuvant (CFA) (Sigma). The mice were injected on days 0, 14 and 28 with 10 μg of cruzipain in 0.1 ml of PBS, mixed with 0.1 ml of CFA 12. Control mice were injected with 0.2 ml of OVA (10 μg) + CFA following the same procedure. Mice were maintained according to National Research Council's guide for the care and use of laboratory animals.

4.3 Purification of mouse IgG

Sera pooled from 10 immunized mice and sera pooled from 10 control mice were obtained 14 days after the third immunization; immune and control pools were then dialyzed overnight against the elution buffer (PBS, pH 8.0). Immune and control IgG fractions were isolated by affinity chromatography using two protein-G–sepharose columns (Amersham Pharmacia) that were previously equilibrated with elution buffer. Immune and control IgG fractions were eluted with 0.1 M citric acid, pH 3.0, neutralized with 2 M Tris and finally they were dialyzed against PBS, pH 7.3. The degree of IgG purification was tested by SDS-PAGE and its concentration was determined.

4.4 M₂ mAChR synthetic peptide

A 24-mer M₂ peptide (VRTVEDGECYIQFFSNAAVTFGTA), corresponding to the sequence of the second extracellular loop of the human mAChR (residues 169–192), was synthesized by the F-moc-amino acids activated using HOBt/DCC strategy. An automatic peptide-synthesizer (Applied Biosystems Model 431A) was used throughout.

4.5 Purification of anti-peptide IgG by affinity chromatography

IgG fraction pools were independently subjected to affinity chromatography on the synthesized peptide covalently linked to AffiGel 15 gel (Bio-Rad, Richmond, CA, USA). The IgG fraction was loaded on the affinity column equilibrated with PBS and the fraction that did not bind peptide was first eluted with the same buffer. Specific anti-peptide autoantibodies were then eluted with 3 M KSCNand 1 M NaCl followed by immediate extensive dialysis against PBS. The IgG concentration of both antibodies that did not bind peptide and specific antibodies that bound the muscarinic-receptor peptide was determined by radial immunodifussion assay and their immunological reactivity against the M₂ mAChR peptide was evaluated by enzyme immunoassay.

4.6 Preparation of membranes

Hearts were removed and placed on a glass plate containing a modified Kreb's-Ringer's bicarbonate (KRB) buffer, gassed with 5% CO₂ in oxygen, pH 7.4; thus, fat, the large vessels, the connective tissue and blood were eliminated. Homogenization was accomplished at 4°C by homogenizing the tissue twice in four volumes of cold buffer containing 0.25 M sucrose, 50 mM Tris-HCl, pH 7.4, and 10 mM MgCl₂ using a Polytron PT-20 at a setting of three for 15 sec. The homogenates were filtered through four layers of gauze and centrifuged at 700×g for 15 min, 10,000×g for 15 min and 40,000×g for 30 min. The pellet was resuspended in 2.5 ml of 50 mM Tris-HCl, pH 7.4, and 10 mM MgCl₂.

4.7 Binding assay

IgG preparations were preincubated with cardiac membranes (3–5 mg/ml protein) for 3 min at 30°C in 5 mM Tris-HCl, pH 7.4, and 10 mM MgCl₂. The cardiac membranes were washed twice by centrifugation and resuspended. For [³H]-QNB binding, 100 μl of membrane suspension (150 μg protein) was incubated with a fixed (0.3 nM) or increasing concentrations of [³H]-QNB (Dupont/New England Nuclear, Boston, MA, USA; specific activity 40 Ci/mmol) in a shaking bath at 30°C for 30 min in a total volume of 150 μl of 50 mM Tris-HCl, pH 8, and 10 mM MgCl₂. In all cases, binding was stopped by the addition of ice-cold buffer and, after filtration under mild pressure, Whatman GF/c filters were rinsed and counted in a Beckman spectrometer. Nonspecific binding was determined by filtering aliquots incubated in the presence of 1 μM atropine, which did not exceed 15% of the specific binding.

4.8 ELISA

A 50-μl volume of the M₂e2 peptide (20 μg/ml), in 0.1 M Na₂CO₃ buffer, pH 9.6, was used to coat microtiter plates (Costar) at 4°C overnight. After blocking the wells with 1% BSA in PBS for 1 h at 37°C, different dilutions of sera or purified IgG from non-infected or infected mice treated or non-treated with M₂e2 (50 μl) were allowed to react with the peptide for 2 h at 37°C 21. The wells were then thoroughly washed with 0.05% Tween-20 in PBS. A 50-μl volume of goat anti-mouse-IgG conjugated to alkaline phosphatase (1:5,000, v/v; Sigma) was added and incubated for 1 h at 37°C. After several washing steps, p-nitrophenyl phosphate (1 mg/ml) was added as substrate and the reaction was stopped at 30 min. values of O.D. were measured with an ELISA reader (Uniskan Laboratory System).

4.9 cGMP and cAMP accumulation assays

Hearts were incubated in 1 ml of KRB for 30 min and different concentrations of IgG preparations or carbachol were added for the last 15 min. When blockers were used, they were added 20 min before the addition of reagents. For cAMP determination, mouse atria were incubated in the presence of 0.1 mM IBMX. To evaluate the inhibitory effects on AMP production, PGE₂ (1×10^–7 M) was added for the last 3 min as an adenylate cyclase stimulator. The residue was dissolved in different buffers for cGMP 6 or cAMP 5. Aliquots of 100 ml were taken for the nucleotide determination using a RIA procedure with [¹²⁵I]cGMP or [¹²⁵I]cAMP RIA kit from Dupont/New England Nuclear, USA.

4.10 Measurements of total labeled IP

Hearts were incubated for 120 min at 37°C in 0.5 ml of KRB (gassed with 5% CO₂ in oxygen) with 1 mCi [³H]MI, specific activity 46.0 Ci/mmol, from Dupont/New England Nuclear; LiCl (10 mmol/l) was added for measurement of inositol monophosphate accumulation, as previously described 10. Carbachol and IgG preparations were added 15 min before the end of incubation period and blockers were added 30 min before addition of reagents. After incubation, atria were quickly washed with KRB and water-soluble IP were extracted. Briefly, tissues were homogenized in 0.3 ml of KRB with 10 mmol/l LiCl and 2 ml chloroform:methanol (1:2, v/v) to stop the reaction. Chloroform (0.62 ml) and water (1 ml) were then added. Samples were centrifuged at 2,000×g for 15 min and the aqueous phase of the supernatants (1–2 ml) was applied to a 0.7 ml column of Bio-Rad AG 1 × 8 anion exchange resin, which had been previously washed with 10 mmol/l Tris-formate, pH 7.4. The resin was then washed with 20 volumes of 5 mmol/l MI to get the first peak of [³H]MI incorporated in the tissue, followed by six volumes of water and, finally, IP were eluted with 1 mol/l ammonium formate in 0.1 mol/l formic acid (second peak). Fractions of 1 ml were recovered and radioactivity was determined by scintillation. Results corresponding to the second peak were expressed as the absolute values of the Simpson's equation. To determine the absence of [³H]MI in the second peak corresponding to IP, thin-layer chromatography on silica gel 60F 254 sheets (Merck, USA) was performed using appropriate MI and IP standards as described previously 6.

4.11 Determination of NOS activity

NOS activity was measured on mouse heart by production of [U-¹⁴C]citrulline from [U-¹⁴C]arginine according to the procedure described previously 6. Briefly, after a 20-min preincubation in KRB 18, tissues were transferred to 500 ml of prewarmed KRB equilibrated with 5% CO2 in oxygen in the presence of [U-¹⁴C]arginine (0.5 μCi). Appropriate concentrations of enzymatic inhibitors were added and tissues were incubated for 20 min, and for an additional 15 min with carbachol or IgG under carbogen at 37°C. Tissues were then homogenized with an Ultraturrax in 1 ml of medium containing 20 mmol/l Hepes, pH 7.4, 0.5 mmol/l EGTA, 0.5 mmol/l EDTA, 1 mmol/l dithiothreitol, 1 mmol/l leupeptin and 0.2 mmol/l PMSF at 4°C. After centrifugation at 2000×g for 10 min at 4°C, supernatants were applied to 2-ml columns of Dowex AG-50 WX-8 (sodium form); [U-¹⁴C]citrulline was eluted with 3 ml of water and quantified by liquid scintillation counting. Measurement of basal NOS activity by the above-mentioned procedure was inhibited to a 95% by 0.5 mmol/l L-NMMA. In order not to drastically modify basal NOS activity, we chose 10 μmol/l L-NMMA to inhibit NOS activation. The activity of the partially purified enzyme was 345+35 nmol/mg/min, as assessed by means of 2′,5′-ADP-Sepharose chromatography. The Ca²⁺-dependency of NOS was assayed in the presence of 1 mmol/l EDTA and free Ca²⁺ was controlled by the addition of CaCl2 solution to the reaction mixture. In the absence of free Ca²⁺ (1 mmol/l EDTA) there was no measurable formation of NO, and a 50% of maximal stimulation of enzyme activity was observed with 0.5 mmol/l of free Ca²⁺.

4.12 mAChR functional studies

Animals were decapitated and the atria were removed quickly and placed in a glass chamber containing KRB solution, pH 7.4, that was gassed with 5% CO₂ in oxygen at 30°C. After a stabilization period of 30 min, spontaneous tension and frequency were recorded using a force transducer coupled to an ink-writing oscillograph, as previously described 6. Then, the preparations were paced by means of a bipolar electrode using a SK4 Grass stimulator, with stimuli duration of 2 msec and a voltage that was 10% above threshold. The constant resting tension applied to the atria (preload tension) was 350 mg. Inotropic effects (dF/dt) were assessed by recording the maximum rate of tension development during electrical stimulation at a frequency of 250 beats/min. To obtain the maximum serum or IgG effect, different concentrations of sera or IgG were added to normal mouse atria every 15 min.

4.13 Drugs

Carbachol, AF-X116, RG E2, L-NMMA and db-cAMP were from the Sigma Chemical Company (Saint Louis, MO), and U-73122 and ODQ were from Tocris Cookson Inc (Ellisville, MO).

4.14 Statistical analysis

Statistical significance was determined by the two-tailed t-test for independent populations. Analysis of variance and Student-Newman-Keuls test was employed when multiple comparisonswere necessary. Differences between means were considered significant if p was equal to or less than 0.05.