Cardiovascular Research

Cardiovascular Research 1999 42(3):685-695; doi:10.1016/S0008-6363(99)00012-7
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Myocardial injury leads to a release of heat shock protein (hsp) 60 and a suppression of the anti-hsp65 immune response

Georg Schett^1,a, Bernhard Metzler^a, Roman Kleindienst,^a,b, Albert Amberger^a, Heidrun Recheis^c, Qingbo Xu^a and Georg Wick^a,c,*

^aInstitute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria
^bDepartment of Internal Medicine, University of Innsbruck, Medical School, Innsbruck, Austria
^cInstitute for General and Experimental Pathology, University of Innsbruck, Medical School, Innsbruck, Austria

^* Corresponding author. Tel.: +43-512-583-9190; fax: +43-512-583-9198

Received 2 September 1998; accepted 13 November 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: While atherosclerosis is associated with high titers of autoantibodies to bacterial hsp65 crossreacting with human hsp60 (anti-hsp60 autoantibodies), myocardial infarction entails decreased humoral immune response to hsp65. We previously hypothesized that myocardial ischemia and subsequent infarction not only induce myocardial hsp60 expression, but also trigger release of myocardial hsp60 into the circulation, influencing the systemic hsp immune response via immune complex formation. Methods: In the present study, organ culture of rat hearts under circulatory arrest provided a model of myocardiocyte injury due to ischemia. Results: Reperfusion of ischemic hearts confirmed the occurrence of myocardial injury by a rise of heart enzymes. Myocardial hsp60 expression was induced up to threefold in response to ischemia, and most of hsp60 expression was localized to the muscle fibers. Analysis of coronary eluate revealed release of hsp60 from myocardium. In addition, hsp60-containing, but not hsp60-free, coronary eluate was recognized by anti-hsp65 serum antibodies and induced proliferation of hsp65-specific T cells. When hsp60-containing coronary eluate was reinjected into an hsp65-primed rat, both humoral and cellular hsp65-immune responses were strongly downregulated. Conclusion: Our findings demonstrate the release of highly immunogenic and crossreactive hsp60 into the circulation in response to myocardial ischemia and myocardiocyte injury.

KEYWORDS Immunology; Infarction; Ischemia; Myocardiocytes; Reperfusion; Heat shock proteins

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Stress proteins of the heat shock protein (hsp) 60 family are among the most potent immunogens, playing a pivotal role in antimicrobial immunity as well as furnishing a link to autoimmunity, as illustrated by the presence of an anti-hsp60 immune response in a number of autoimmune diseases and atherosclerosis [1–4]. A number of independent groups have uncovered cardivascular risk factors, including hypertension [5,6] and oxidized lipoproteins [7–9], as inducers of hsp responses. Furthermore, immunologic and autoimmunologic reactivity to hsp60 of both the humoral and cellular types have been shown to play an initiating role in atherogenesis, reflecting a strong link between vascular stress protein expression, vascular pathology, and the hsp60 immune response. For example, serum anti-hsp65/60 antibodies with cytotoxic activity against endothelial cells were found in high levels in the presence of carotid or coronary atherosclerosis [10,11].

Paradoxically, studies investigating the humoral immune response against hsp65 in the presence of myocardial injury reported decreased hsp-antibody titers following myocardial infarction, despite the presence of atherosclerotic lesions [12,13]. The hypothesis was that myocardial injury induced hsp60 release into the circulation, which interacted with the anti-hsp65 immune response based on its high sequence homology, leading to immune complex formation.

Since the hsp60 molecule is known as a predominantly intracellularly-located protein, the possibility of interaction with the immune system is controversial [14]. Cell death, especially necrosis, may lead to the liberation of considerable amounts of hsp60 into the circulation, rendering it accessible to the immune system and decisively influencing the anti-hsp immune reaction. Surface expression [15–20], and release [21,22], of hsp60 provides another mechanism of immune system interaction and has been described in association with cell stress and cellular apoptosis [23].

Whether stress proteins are released during myocardial injury and, if so, what their impact might be on an anti-hsp immune response had not been previously determined. In the present study, we investigated whether (a) myocardial ischemia induces myocardial hsp60 expression, (b) myocardiocyte injury induces hsp60 release into the circulation, (c) released myocardial hsp60 was recognized by an hsp65-specific immune response, and (d) released myocardial hsp60 influenced an hsp65-specific immune response.

Experiments were performed using an ex vivo rat heart ischemia model similar to the Langendorff perfused heart with different times of circulation arrest in organ culture and subsequent reperfusion. This model does not investigate the recovery from circulatory arrest or the myocardial effects of reperfusion, as in the classical Langendorff perfused heart, but uses myocardial ischemia and subsequent myocardiocyte injury due to circulatory arrest as a basis to study the kinetics of hsp60 expression and release, as well as the systemic consequences of reperfusion of damaged and/or infarcted tissue. Therefore, prolonged periods of circulatory arrest were also investigated.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Animals and organ culture
Six week old female Lewis rats kept at 25°C and 95% humidity were fed a standard diet and received water ad libitum. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). Thirty min before organ explantation, anticoagulation was performed by intravenous injection of 200 I.U. preservation free heparin (Immuno, Vienna, Austria) into the tail vein. Preliminary results did not show any influence of an in vivo administration of heparin on the experiments performed, however the initial ex vivo-reperfusion of hearts was strongly facilitated. Hearts were then explanted via sternotomy in ether anaesthesia and transferred to 37°C-prewarmed medium (RPMI 7640 supplemented with 1% rat serum, 100 U/ml L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin). Aortae were prepared according to the technique of the Langendorff perfused heart and permanently cannulized by a pipet tip secured with silk sutures. Coronary arteries were RPMI-perfused by connecting the aortal pipet tip to a 10 ml syringe, and an initial perfusion of 20 ml prewarmed RPMI was carried out to remove remaining blood cells from the coronary circulation. Hearts were subsequently transferred to organ culture with 10% fetal calf serum (FCS, Seromed, Berlin, Germany; shown to be free of hsp60)/RPMI in a six-well plastic plate (Costar, Cambridge, MA, USA) and incubated at 37°C and 5% CO₂ for various periods of circulatory arrest. Thereafter, further perfusions with 1 ml RPMI were performed, and the coronary eluate analyzed with respect to the various parameters. Each eluate was filtered through a 0.2 µm millipore filter (Sartorius, Göttingen, Germany) to remove any intact cell or cell debris.

2.2 Enzyme kinetics
To confirm myocardiocyte injury the levels of "heart enzymes", including creatine kinase (CK) and lactate dehydrogenase (LDH), as well as levels of troponin T, were measured in the coronary eluate at each time interval of ischemia. CK was assessed by photometric measurement of NADP reduction at 334 nm, LDH by photometric measurement of NADH decrease at 334 nm, and troponin T by immunoassay [24]. All measurements were performed at the Department of Laboratory Medicine of the Innsbruck University, Clinic for Internal Medicine.

2.3 Sodium dodecyl sulphate polyacrylaminde gel electrophoresis (SDS-PAGE) and Western blotting
Immunoblotting was used to analyze coronary eluate for the presence of hsp60. Briefly, eluates (100 µg/lane) were diluted 1:2 (v/v) with electrophoresis sample buffer (5% β-mercaptoethanol, 15% glycerol, 3% SDS, 0.1 M Tris, pH 6.8) and subsequently boiled at 97°C for 5 min. Probes (5 µl eluate) were then resolved on 12% polyacrylamide gels under reducing conditions and proteins electrophoretically blotted onto nitrocellulose membranes (BA85; Schleicher und Schuell, Dassel, Germany) in 25mM Tris, 192 mM glycine, 20% methanol, pH 8.3, at 100 mA for 16 h. After blocking the membranes with 3% nonfat dry milk/phosphate buffered saline (PBS), pH 7.2 (Sigma, Munich, Germany) for 1 h, blots were probed with a monoclonal antibody to mammalian hsp60 (clone II-13, recognizing aa288-366; a gift from R.S. Gupta, Hamilton, Canada) for another hour. The reaction was visualized using horse radish peroxidase (HRP)-conjugated rabbit anti-mouse Ig secondary antibodies (P260, Dako, Copenhagen, Denmark) followed by addition of its substrate 4-chloro-1-naphtol/hydrogen peroxide (Sigma).

When investigating hsp60 and hsp70 expression in the myocardium, hearts recovered after certain periods of ischemia were homogenized in a homogenizer (Polytron) in lysis buffer containing 0.15 M NaCl, 50 mM Tris, 1 mM EDTA, 0.05% SDS, 0.5% Triton X100 and 1 mM phenylmethylsulfonylfluoride (pH 7.4). The supernatant was harvested and the protein content measured by Bradford-assay (Bio-Rad, Hercules, CA, USA). Each lane was loaded with 100 µg myocardial protein. A monoclonal antibody to mammalian hsp70 (clone W28, StressGen Biotechnologies, Victoria, BC, Canada) was used to detect that stress protein.

2.4 Enzyme-linked immunosorbent assay (ELISA)
The release of hsp60 was quantified in coronary eluate by a Sandwich-ELISA with two monoclonal antibodies against mammalian hsp60. Briefly, ELISA plates (11041E, PetraPlastik, Würzburg, Germany) were coated with 100 µl of the Mab II-13 (dil.: 1:4000) at 4°C overnight. After washing with PBS supplemented with Tween 20 (0.05%, v/v) and blocking with 5% nonfat milk, 100 µl of appropriately diluted coronary eluate (dil. 1:20) were added for 1 h. As a second hsp60-specific monoclonal antibody, 100 µl of biotinylated Mab ML-30 (dil.: 1:250, recognizing epitope aa315-318 common to mycobacterial hsp65 and human hsp60; a gift from Dr. Ivanyi, London, UK) was used and the reaction visualized by the addition of peroxidase-conjugated streptavidin (P397, Dako, dil.: 1:5000) and its substrate, 2,2'-azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS; Sigma). Absorbance was measured at 410 nm with a Microelisa Autoreader (Dynatech, Alexandria, VA, USA). A standard extinction curve was drawn by simultaneous determination of OD-values of serially-diluted recombinant mammalian hsp60 (5 to 1000 ng/well; Stressgen) in the ELISA described above. The OD-value of each coronary eluate was referred to this standard extinction curve to quantify hsp60 content. In each experiment, samples (coronary eluate), serially-diluted recombinant hsp60 and negative controls (1 µg ovalbumin/well) were tested on the same plate. Test specificity was confirmed by blocking with 100-fold excess of a 15-mer peptide of hsp60 (aa310-325; a gift from Dr. Ottenhoff, Department of Immunohematology and Blood Bank, Leiden, The Netherlands [25]) specific for the ML-30 Mab and the same amount of a control peptide (aa320-335). For additional control purposes, hsp60-containing eluate (200 µl) was mixed with 20 µl of Mab II-13, Mab ML-30, or control Mab anti-rat CD3 (M756, Dakopatts) for 1 h at room temperature, followed by addition of 50 µl protein A-sepharose beads for 4 h. Bound antibodies were then removed by centrifugation (12 000 g, 30 s.) and supernatants analyzed by ELISA as described above.

2.5 Immunofluorescence
To investigate the induction and localization of myocardial hsp60 expression, immunofluorescence-staining of 7 µm-thick serial cryosections of myocardial tissue blocks frozen in liquid nitrogen was performed. Sections were fixed with acetone for 10 min, transferred into a humidified chamber and incubated with a specific Mab to hsp60 (II-13, dil.: 1:250) for 30 min. After washing in PBS, bound antibody was detected by 30 min incubation with a fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin (P261, Dako, dil.: 1:50). Counterstaining of endothelium by a tetramethylrhodamine isothiocyanate (TRITC)-labeled lectin of Bandereia simplicifolia-BS I (dil:: 1:10, L5264, Sigma) to visualize coronary vessel endothelial cells was performed after staining with hsp60-specific Mab. Thoroughly washed sections were embedded in Mowiol (Sigma) and immediately examined with a laser scanning microscope LSM 10 (Zeiss, Germany). Digital images of fluorescence in response to excitation with an argon laser (488 nm), filter setting BP 530/30 for FITC-staining, and with the helium neon laser (543 nm), filter setting BP 575-640 for TRITC staining, were collected at a scan rate of 2 s per image. Digital images were stored on a hard disk, transferred to a high resolution RGB color video photomonitor (Lucius and Baer, Geretsried, Germany), and photographed using Fujicolor Super HG 200 films. Quantification of fluorescence intensity was computed by measuring the fluorescence intensity at three independent areas of each section, representing localizations with high, average and low fluorescence intensity and formation of a mean value of these three measurements.

2.6 T cell culture and proliferation
Specific antibodies and T cells against hsp65 were isolated from peripheral blood of 8 week old female Lewis rats immunized with 100 µg recombinant hsp65 in Freunds incomplete adjuvant into one hind food pad. After 10 days, the animals were sacrificed and serum and lymphocytes from the draining popliteal lymph nodes collected. Based on a previously-described protocol [26], specific T cell lines against hsp65 were grown by stimulation with recombinant hsp65 (20 µg/ml) in the presence of a 50-fold excess of autologous irradiated thymic feeder cells. Cell culture was performed using DMEM supplemented with 1% autologous rat serum, 2-mercaptoethanol (5x10^–5 M), nonessential amino acids, sodium pyruvate and antibiotics. Propagation of T cell lines was achieved by cultivation in 10% FCS/DMEM supplemented with 15% (v/v) conditioned medium (supernatant of concanavalin A [ConA]-stimulated spleen cells). Proliferation tests were performed by pulsing 1x10⁴ T cells with 5 µg recombinant antigen (hsp65, hsp60, groEL: StressGen; ovalbumin, bovine serum albumin: Sigma; purified protein derivative [PPD]: Statens Seruminstitut, Copenhagen, Denmark) or, alternatively, 20 µl of coronary eluate in the presence of 1 µ Ci ³H-thymidine/well for 72 h.

For testing the in vivo effect of myocardial infarction on the hsp65 response, 10 µl coronary eluate were injected intravenously into hsp65-primed rats. After 2 weeks the animals were killed, serum analyzed for hsp65 antibodies, and peripheral blood mononuclear cells (PBMC) as well as spleen cells tested for their proliferative response to hsp65. Proliferation tests were performed as described above. Absorption of hsp60 from hsp60-containing coronary eluate (24 h) was performed by addition of 20 µg/ml Ab II-13 and precipitation with protein A agarose beads. Absorbed (abs, hsp60 free) and immunoprecipitated (ip, hsp60 containing) fractions were reinjected.

2.7 Serum anti-hsp65 antibodies
An ELISA following an established protocol [10] was used to identify hsp65 antibodies. Briefly, 96-well flat-bottom ELISA plates (11041E, PetraPlastik) coated with 1 µg/ml recombinant hsp65 were incubated with 100 µl of hsp65-immunized or nonimmunized rat serum. As detection antibody, a HRP-conjugated rabbit anti-rat Ig (Z494; Dako) was used. The reaction was visualized with the ABTS substrate and absorbance measured at 410 nm with a Microelisa Autoreader.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Enzyme kinetics
The levels of troponin T, CK and LDH in coronary eluate were measured at each ischemic time point. Initial perfusion (0 h) and the first reperfusion (2 h) of the explanted heart induced no detectable rise in heart enzymes, reflecting an intact myocardium (Fig. 1). However, after 6 h of organ culture, CK, LDH and troponin T showed a weak, but significant, rise, reflecting myocardiocyte injury. Maximum enzyme release was detected after 24 h in the case of troponin T and CK, and between 12 and 24 h in the case of LDH. The levels of all three parameters decreased after 36 h of organ culture, but were still detectable at high levels.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Levels of heart enzymes and troponin T. Hearts were kept in organ culture in 10% FCS/RPMI at 37°C in humidified atmosphere containing 5% CO2. After various periods of circulation arrest, reperfusion was performed and the eluate quantitatively analyzed for the levels of troponin T, CK and LDH. Measurements were performed at the Department of Laboratory Medicine at the University of Innsbruck, Medical School. Values (troponin T: ng/ml; CK and LDH: U/l) are means of three independent experiments, the arrows indicate the start of a significant increase.

 
3.2 Release of myocardial hsp60
Quantitative assessment of hsp60 release was performed by Sandwich-ELISA using two anti-hsp60 Mabs and serially-diluted recombinant rat hsp60 as a reference (Fig. 2). The initial perfusate (0 h) was completely free of hsp60, the first reperfusion (2 h) showed minimal hsp60 release (0.075 µg/ml), whereas prolonged ischemia resulted in a strong increase of hsp60 release detected in the coronary eluate after 6 h (1.86 µg/ml), 12 h (5.10 µg/ml) and 24 h (11.59 µg/ml). As demonstrated by Western blotting, the levels of released protein decreased after 36 h of organ culture (6.40 µg/ml). When correlating hsp60 release to myocardial tissue weight (g myocardium), the released hsp60 quantities were 0 µg/g (0 h), 0.062 µg/g (2 h), 1.55 µg/g (6 h), 4.25 µg/g (12 h), 9.66 µg/g (24 h) and 5.33 µg/g (36 h). Specificity was confirmed by almost complete inhibition when hsp60-peptide aa310-325 (including the epitope recognized by Mab ML-30) was added to the test, but not when adding the control peptide aa320-335. Furthermore, depletion of hsp60 from the eluate by immunoprecipitation with Mab II-13 or Mab ML-30 completely blocked the Sandwich-ELISA, whereas a nonrelated antibody (anti-rat CD3) had no effect (Fig. 3).

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Quantification of hsp60 release. Quantitative assessment of hsp60 in coronary eluate was performed by a Sandwich-ELISA. Microtiter ELISA-plates, coated with hsp60-specific Mab II-13 (dil.: 1:4000), were blocked and diluted coronary eluate from 0–36 h of organ culture (dil.: 1:20), as well as serially diluted recombinant rat hsp60 (5–1000 ng/well), were added for 1 h. Detection of bound hsp60 was performed by using a second biotinylated hsp60-specific Mab ML-30 (dil.: 1:250) and streptavidin–peroxidase (dil.: 1:5000). Values are means of three independent experiments, the x-axis indicates the duration of organ culture, the y-axis the concentrations of released coronary hsp60 (µg/ml).

 

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Specificity of hsp60-ELISA. Specificity of hsp60 recognition by the ELISA described above was proven by blocking. Briefly, an anti-hsp60 Mab (II-13; dil.: 1:4000) was used as a capture antibody for recombinant hsp60 (50 ng, column 1) and coronary eluate of 0 h (dil.: 1:20, 2) and 24 h (dil.: 1:20, 3-8). After incubation with a second biotinylated anti-hsp60 Mab (ML-30, dil.: 1:250), the reaction was visualized by streptavidin–peroxidase. Blocking was performed by addition of 100-fold excess of hsp60-peptides aa310-325 (5 µg, 4) and aa320-335 (5 µg, 5). In addition, 24 h-coronary eluate was tested after precipitation with protein A sepharose-bound Mab II-13 [6], Mab ML-30 [7] and Mab anti-CD3 [8]. Values indicate mean optical densities at 410 nm. Data of two independent experiments performed in triplicate.

 
The presence of hsp60 in coronary eluate was also investigated by immunoblotting of released myocardial proteins separated by SDS-PAGE (data not shown). Coronary eluates after 0 and 2 h of organ culture lacked detectable hsp60, ruling out the presence of hsp60 from remaining blood cells in the coronary circulation. After 6 h a release of hsp60 into the coronary circulation was detected, strongly increased up to 24 h of ischemia, and thereafter showed a small decrease (36 h).

3.3 Myocardial hsp60 expression
At each ischemia timepoint Western blot analysis of myocardial hsp60 expression was performed using 50 µg blotted myocardial protein derived from homogenized heart tissue. Normal myocardium revealed a low, but detectable, baseline expression of hsp60 that remained unchanged during the first 2 h of ischemia (Fig 4). Increased myocardial hsp60 expression was observed at 6 h of organ culture, with kinetics similar to hsp60 release into the coronary circulation. This expression remained high up to 36 h of ischemia. Densitometric evaluation indicated an up to threefold increase in expression following ischemia.

View larger version (46K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Immunoblot of myocardial hsp expression. Hearts were kept for various times (0–24 h) in organ culture. After homogenization of myocardial tissue in lysis buffer and subsequent protein extraction, 100 µg/lane myocardial protein at each timepoint of ischemia, diluted 1:2 in electrophoresis buffer, was separated on a 12% SDS-PAGE under reducing conditions. After blotting proteins onto nitrocellulose membrane immunodetection with the hsp60-specific Mab II-13 (upper panel) or an hsp70-specific Mab (lower panel) was performed. The expression of hsp60 and hsp70 after 0 h, 2 h, 6 h, 12 h and 24 h of circulation arrest in organ culture is shown.

 
Immunofluorescent staining of hsp60 on cryosections of myocardial tissue confirmed increased expression in response to ischemia (Fig.5). In particular, intensive staining was observed after 12 and 24 h of ischemia. Semiquantitative assessment of fluorescence intensity by confocal microscopic measurements at three independent locations revealed a twofold increase of staining intensity (data not shown). Most hsp60 staining was located within the myocardial cells, and far less within the coronary vessel walls, as demonstrated by counterstaining with an endothelial-specific marker (Fig. 5 E,F).

View larger version (114K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Immunofluorescence of myocardial hsp60 expression. Acetone-fixed, 7 µm cryosections of heart tissues after various times of circulation arrest in organ culture were analyzed for the expression of hsp60. Immunodetection of hsp60 was performed by incubation with hsp60-specific Mab II-13 (dil.: 1: 200) and visualization by FITC-conjugated anti-mouse Ig (dil.: 1:50). Sections were embedded in Mowiol and analyzed by laser confocal microscopy. Pictures show (A) myocardial tissue after 0 h, (B) after 6 h, (C, E, F) after 12 h and (D) after 24 h of organ culture. Counterstaining of endothelial cells (E, F) was done by TRITC-labeled Bandereia simplicifolia lectin (dil.: 1:10) for the visualization of coronary vessels.

 
3.4 Immunologic recognition of released hsp60
To investigate possible immunologic recognition of released myocardial hsp60, anti-hsp65 high titer antiserum was tested for binding and an hsp65-specific T cell line for proliferation to hsp60 present in the coronary eluate. Anti-hsp65 high titer antiserum from rats immunized with recombinant M.tb. hsp65 exhibited an hsp65-antibody titer of more than 1:10000, as determined by ELISA. High titer antiserum was proven to crossreact with recombinant rat hsp60 (Fig. 6a, lane A), as well as with released myocardial hsp60 in the coronary eluate of 24 h of organ culture (lane C), but not eluate devoid of hsp60 (0 h, lane E). Normal rat serum showed no reaction (lanes B, D, F).

View larger version (32K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Recognition of released myocardial hsp60 by specific anti-hsp65 antibodies and T cells. (a) Recombinant rat hsp60 (A,B, 1 µg/lane), hsp60-containing coronary eluate (CE 24) of 24 h ischemic organ culture (C,D, 50 µl/lane) and hsp60-free coronary eluate (CE 2) of 2 h of organ culture (E,F, 50 µl/lane) were diluted 1:2 in electrophoresis buffer and separated in a 12% SDS-PAGE under reducing conditions. Proteins were blotted onto nitrocellulose membranes and blots probed by rat anti-hsp65 high titer antiserum (A,C,E; dil.: 1:200) or normal rat serum (B,D,F; dil.: 1:200). The reaction was visualized by incubation with HRP-conjugated anti-rat Ig and its substrate. Note the 60-kD band with anti-hsp65 high titer antiserum in lanes A and C. (b) 1x104 cells of an hsp65-specific T cell line were cultivated in the presence of antigen and 5x105 autologous irradiated thymic feeder cells for 72 h and pulsed with 1 µCi 3H-thymidine/well for the last 24 h. 3H-thymidine incorporation was measured in a beta-counter and specific proliferative activity was computed by subtraction of unspecific proliferation in the absence of antigen but presence of feeder cells. The following antigens were used: M.tb. hsp65 (10 µg/well), rat hsp60 (10 µg/well), E. coli groEL (10 µg/well), M.tb. purified protein derivative (PPD, 10 µg/well), bovine serum albumin (BSA, 10 µg/well), ovalbumin (OA, 10 µg/well), 2 h-coronary eluate (20 µl), 24 h-coronary eluate (20 µl) and concanavalin A (ConA, 2 µg/well). Values of specific proliferation are means of three independent experiments.

 
The hsp65-specific T cell line was grown by repeated (five cycles) restimulation with recombinant hsp65 and presented a specific reaction with hsp65, as reflected by nearly identical proliferation to hsp65 and to the mitogen ConA (Fig. 6b). In contrast, no proliferation was observed to ovalbumin or bovine serum albumin, and only minor proliferation to groEL, the hsp60 analogue of E. coli. While proliferation to hsp65-containing M.tb. PPD was expected, the T cell line gave a surprisingly high response to recombinant rat hsp60, and to a lesser extent, to released myocardial hsp60. Again, no effect was observed when hsp60-free coronary eluate was added to the culture.

3.5 Effect of released hsp60 on the hsp65 immune response
To elucidate whether and how released myocardial hsp60 influences a preexisting hsp65-specific immune response, intravenous reinjection of hsp60-containing (CE 24h) and hsp60-free (CE 2h) coronary eluate into hsp65-primed rats was carried out. Unprimed rats showed only low hsp65-specific T cell and antibody responses (Tables 1 and 2), whereas immunization with recombinant hsp65 resulted in a strong induction of a specific antibody and T cell response. Induction of an hsp65-directed T cell response was evident both in the peripheral blood and spleen. Following injection of hsp60-containing coronary eluate into hsp65-primed rats (hsp65+CE 24h), both peripheral blood and splenic hsp65-T cell responses were markedly suppressed, although not to baseline levels, and the hsp65 antibody response was significantly diminished. These phenomena did not occur in response to hsp60-free coronary eluate (hsp65+CE 2h), maintaining hsp65 T cell and antibody responses at the level of hsp65-immunized animals. Preabsorption of of hsp60-containing coronary eluate with anti-hsp60 monoclonal antibody did abolish the suppressive effect on hsp65 antibody and T cell responses (hsp65+CE 24h/abs, Tables 1 and 2). However, immunopurified hsp60 from coronary eluate (hsp65+CE 24h/ip) was suppressive.

View this table: [in this window] [in a new window]   Table 1 Effect of reinjection of myocardial hsp60 on the systemic hsp65-antibody response

 

View this table: [in this window] [in a new window]   Table 2 Effect of reinjection of myocardial hsp60 on the systemic hsp65-T cell response

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Herein, we demonstrate in an ex vivo-rat heart ischemia model that myocardiocyte injury is paralleled by a release of hsp60 into the coronaries, and thus systemic circulation. The presence of hsp60 in the coronary eluate was confirmed by the following experiments: (1) the presence of a 60-kD band when probing blotted coronary eluate by a hsp60-specific Mab; (2) detection and quantification of hsp60 in the coronary eluate by Sandwich-ELISA using two Mabs specific for hsp60; (3) recognition of released protein by a high-titer anti-hsp65 antiserum, and (4) the recognition of released protein by a hsp65-specific T cell line. Release of myocardial hsp60 is accompanied by an upregulation of myocardial hsp60 expression in response to ischemia, and induces suppression of the humoral and cellular immune response against hsp65.

Myocardial ischemia is a known potent inducer of the stress protein response. Several stimuli trigger upregulation of myocardial stress proteins in response to ischemia, such as hypoxia [27,28], decreased energy storage, particularly ATP levels [29], acidic pH [30], presence of denatured proteins [31], and oxidative stress [32–34]. Induction of the heat shock response supports the survival of myocardial cells [29], and numerous studies have reported that induction of hsp70 in myocardial cells enhances myocardial survival during ischemia and reduces infarct size [35–37]. In contrast, little is known about the hsp60 response, although recent studies show upregulation of myocardial hsp60 expression in response to ischemia [38,39] suggesting signalling via adenosin receptor activation [38] as well as a protective effect of hsp60 overexpression from ischemia-induced injury [40]. In our model, upregulation of hsp60 up to threefold was observed, with onset of protein increase (6 h of ischemia) concomitant with both myocardial injury, as seen from increased heart enzyme levels, and with release of hsp60 into circulation. hsp60 Induction is quickly regulated and may be a response to a rapid requirement of hsp60 due to a fast decline of cell metabolism, which renders transcriptional regulation inadequate. In lower concentrations, hsp60 was also detectable in unaffected and briefly ischemic myocardium, indicating its essential role in normal organ function. Heart hsp60 and hsp60 upregulation are largely observed within the myocardiocyte, as shown by immunofluorescence, with lesser amounts being found in coronary vessel walls. This distribution may be due to the predominantly mitochondrial localization of hsp60 and the abundance of mitochondria in the muscle fibers. In contrast, when analyzing hsp70 in myocardial tissue, no expression was detected in unaffected and briefly ischemic hearts. Induction of expression in ischemic hearts commenced at 6 h of ischemia with kinetics similar to hsp60.

Release of hsp60 into the coronary arteries was concomitant with myocardial upregulation and increased with longer periods of ischemia. Most of the released hsp60 may result from myocardial necrosis, since similar kinetics of increase of heart enzymes were observed. In addition, other factors, particularly cell stress and apoptosis, may be important, leading to surface expression of hsp60 in eukaryotic cells [15–20]. Whether surface-hsp60 can be shed from stressed or apoptotic cells remains to be clarified. The observation of normally intracellularly-localized hsp70 in the serum in the presence of myocardial infarction with preceeding angina, but not without preceeding angina, may indicate stress protein release in the absence of necrosis [41].

A number of different autologous proteins are liberated following myocardial injury, but only some of these are immunogenic. The hsp’s of the hsp60 family represent one of the most potent immunogenic protein groups and, due to high interspecies homology and abundancy, crossreactive immune reactions occur [3,42,43]. Here, we demonstrate that released myocardial hsp60 is recognized by a preexisting hsp65 immune response both at the antibody and at the T cell levels, suggesting that the anti-hsp65 immune response is at least partially influenced by the immunologic accessibility of self-hsp60, such as following myocardial infarction. This mechanism may also be important in circumstances where self-hsp60 interacts with the immune system, such as the release of hsp60 as a consequence of necrosis, and surface expression of hsp60 after cell stress. Preexisting cellular and humoral hsp65 immune responses are widespread in laboratory animals and humans and originate from various conditions, including bacterial infection, vaccination, atherosclerosis and autoimmune diseases [44,45]. Depending on epitope recognition, crossreactivity, and other nonimmunologic factors, the anti-hsp65 immune response is beneficial (host defense) or harmful (triggering autoimmune disease) [45].

Crossreactivity of anti-hsp65 serum antibodies with mammalian hsp60 has been repeatedly described and is based on the recognition of conserved homologous epitopes on bacterial and mammalian hsp’s [46]. Since part of the anti-hsp65 antibody response is therefore also directed against autologous hsp60, released self-hsp60 can bind to circulating anti-hsp65-antibodies, and thus lower hsp-antibody titers, as demonstrated by reinjection of released myocardial hsp60. Reinjection of myocardial proteins released following myocardial injury into an healthy animal provides a model to study systemic reactions to released proteins. The massive decrease in antibody titers following reinjection of hsp60 supports the high degree of crossreactivity of anti-hsp65 antibodies. Depending on the in vivo-kinetics of hsp60–anti-hsp65 antibody complex elimination, the decreased antibody titers are based on the rapid clearance of antigen–antibody complexes via the Fc-receptors of the cells of the reticuloendothelial system or, if still circulating, by the failure of the ELISA to detect antigen-bound antibody. In this context, the phenomenon of decreased human anti-hsp65 antibody titers in patients with myocardial infarction deserves mentioning [12,13]. Despite the presence of coronary heart disease and the fact that this disease, like atherosclerosis, is generally accompanied by a rise in anti-hsp65 antibodies, titers paradoxically fall when myocardial infarction occurs. Based on our data, the most likely explanation is a release of myocardial hsp60 during infarction and subsequent binding to, and elimination of, specific antibodies.

In addition to interacting with antibodies, released hsp60 also comes into contact with preexisting antigen-specific T cells. Using an hsp65-specific T cell line, we demonstrated the capacity of released myocardial hsp60 to stimulate and induce proliferation in hsp65-specific T cells. The T cell line tested exhibited a high degree of crossreactivity with mammalian hsp60, and was thus chosen to screen the hsp60-containing eluate for proliferative potential. Of several hsp65-specific cell lines only a minor part showed equal crossreactive potential with mammalian hsp60. Some showed only low activity (data not shown), most likely based on their preferential recognition of nonhomologous epitopes. This is also reflected by the low degree of proliferation in response to homologous E.coli-hsp60, groEL. Surprisingly, reinjection of released hsp60 induced a decreased hsp65 T cell response of both peripheral blood and splenic T cells. The possibility that reinjection of coronary eluate may itself alter the T cell response can be ruled out, because the mitogenic T cell response remained unchanged and coronary eluate without hsp60 had no effect on the hsp65 T cell response. Released myocardial hsp60 may be ingested and processed by circulating and/or resident antigen presenting cells able to stimulate hsp65-specific T cells. Exogenous hsp65, like endogenous hsp60 can also be presented through the MHC-class-I pathway to T cells [47]. The decreased anti-hsp65 T cell response is most likely a decreased CD4-T cell response, since proliferation tests measure the response to an exogenously added antigen presented through the MHC-class-II pathway. Whether the lack of a costimulatory signal and resulting T cell anergy, or induction of T cell suppression, is responsible for decreased hsp65 T cell response following injection of hsp60-containing eluate remains to be clarified. In vivo, the pathophysiologic role of an autoimmune response to hsp60 and subsequent immune complex formation after myocardial infarction may be based on the removal of antigenic material from damaged cells, representing a "housekeeping" function of the immune system. Alternatively, the development of an immune complex disease may be initiated, however its symptoms would be transient because of the limited time of antigen presence.

Further studies investigating antibodies and T cells specific for mammalian hsp60, but not hsp65, should clarify whether the release of larger quantities of hsp60 into the circulation gives rise to a bona fide primary immune reaction to self-hsp60 [4] despite downregulation of the anti-hsp65 response. Demonstrating induction and release of a highly immunogenic and abundant autoantigen, such as hsp60, into the circulation, may advance our understanding of the systemic effects of reperfusion of damaged and/or infarcted tissue on the antigen-primed immune system.

Time for primary review 26 days.

	Acknowledgements

 
This work was supported by the Austrian Science Fund (G.W., project no.12213). We thank Dr. Radhey S. Gupta (Department of Biochemistry, McMaster University, Hamilton, Canada) for kindly providing recombinant rat hsp60 and monoclonal antibody II-13 and Dr. Lee Mizzen (Stressgen Biotechnologies, Victoria, BC, Canada) for providing recombinant mycobacterial hsp65 and E. coli groEL. Furthermore, we are grateful to Dr. Elmar Jarosch (Central Laboratory Unit, University Hospital Innsbruck, Austria) for the quantitative assessment of heart enzymes, to Dr. Jurai Ivanyi (MRC London, UK) for providing the Mab ML-30, to Dr. Tom Ottenhoff for synthetic peptides and to Mr. Thomas Öttl for the preparation of photographs.

	Notes

 
¹ Present adress: Department of Rheumatology, III. Clinic for Internal Medicine of the General University Hospital Vienna, Austria.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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