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Epstein-Barr Virus Mediated Graft Rejection in Heart Transplant Patients: Implication of the Cardiac Cytoskeleton
Epstein-Barr Virus Mediated Graft Rejection in Heart Transplant Patients: Implication of the Cardiac Cytoskeleton M.H. LeBlanc, S. Boudriau, D. Doyle, A. Gagnon, D. Beaudoin, D. Coulombe, O. Gleeton, J.G. Kingma, Jr, and M. Boutet
D
URING the last decade, cardiac transplantation has evolved from experimental procedure to a selected choice for patients suffering from end-stage heart failure. Myocardial ischemia and dilated cardiomyopathy are the most common diseases requiring heart transplantation. However, the success of transplantation is often undermined by serious complications such as graft rejection, infection and neoplasia. Because recipients are treated with high doses of immunosuppressive medication, malignancy develops. One of the most common lymphomas in transplant patients is B-cell associated post-transplant lymphoproliferative disorder (PTLD). Up to 12% of recipients surviving more than 1 month after transplant develop PTLD and, in most cases, there is evidence that EpsteinBarr virus (EBV) may accompany or precede development of the disease.1 In contrast, EBV may be involved in graft rejection by a mechanism involving superantigens. The association of virus with class II major histocompatibility complex (MHC), may induce the formation of superantigens.2 These superantigens are presented to T-lymphocytes in their native form without previous degradation into small peptides. Superantigenic stimulation recruits up to 105-fold more T-lymphocytes compared to conventional antigens, which may induce severe rejection.2 Moreover, when viruses invade cells, they are known to disturb various cellular processes including synthesis and protein assembly.3 In this study, we hypothesized that EBV infecting cardiac myocytes may disrupt cytoskeleton structural proteins, which attach myofibrils to the sarcolemma; this could lead to cardiac contractile dysfunction. The principal objectives were to demonstrate the presence of EBV in cardiac myocytes from heart transplant patients diagnosed with severe graft rejection and extranodal lymphoma and to search for the EBV receptor CD21 and for B-cell activating factor CD23. In a parallel study, we examined the integrity of the cardiac myocyte cytoskeleton in viral infected endomyocardial biopsies to assess the potential relation between viral infection and cardiac myocyte cytoskeleton disruption. MATERIALS AND METHODS Heart Transplant Recipients Twenty-six patients underwent cardiac transplantation between 1993 and 1996. They were all treated with induction therapy 0041-1345/98/$19.00 PII S0041-1345(98)00096-7 918
comprising rabbit anti-thymocyte globulin (RATG) 125 mg (IV) for 3 days followed by standard triple regimen therapy (prednisone, cyclosporine, and azathioprine). Biopsy procedures were performed as part of the routine endomyocardial biopsy (EMB) protocol to monitor patients for acute rejection (weekly for 6 weeks, bimonthly for 2 months and monthly for 6 months). When severe rejection occurred, biopsies were sampled more frequently. EMB were obtained from the right interventricular septum with a bioptome introduced through the right internal jugular vein. Five to six pieces of endomyocardial tissue were obtained during each procedure; one specimen was used for electron microscopy; the others were prepared at random for light microscopy techniques (routine histology, immunohistology and immunofluorescence). Rejection was histologically diagnosed in EMB according to The International Society for Heart and Lung Transplantation (ISHLT) criteria.4 Two patients underwent several episodes of severe rejection and heart failure and developed extranodal lymphoma (PTLD); they died 3 to 6 months after the diagnosis of PTLD. The first patient had a primary lung tumor with secondary involvement of the liver and spleen. The second patient had a primary cardiac tumor at the level of the suture line between the donor and recipient pulmonary artery. Serological tests for cytomegalovirus (CMV) and EBV viral capsid antibody-IgG (VCA-IgG) were done in all these patients5; they were all EBV positive before transplantation; 14 were CMV positive.
Test CMV serological test. This test was performed on whole blood. White blood cells were extracted and concentrated by cytospin. They were stained by immunofluorescence with a monoclonal CMV antibody (Argene-Biosoft, Varilhes, Fr) and positive cells were numbered on a total of 2 3 105 cells. Results were expressed in percentage. EBV serological test. Four different antibodies were detected by titration and immunofluorescence. Tests were considered positive when VCA-IgG and VCA-IgM reached a concentration of 1/100, EBNA 1/10, and EA 1/80 (Caltag Laboratories Inc, San Francisco, Calif).
From the Quebec Heart Institute and Department of Pathology, Laval University, Ste-Foy, Quebec, Canada. Address reprint requests to Dr Marie-H. LeBlanc, Quebec Heart Institute, 2725, Chemin Ste-Foy, Ste-Foy, (Quebec), G1V 4G5, Canada. © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 30, 918–924 (1998)
EPSTEIN-BARR VIRUS MEDIATED GRAFT REJECTION
Rationale Electron microscopy and routine histology were performed after each biopsy procedure in all patients studied. Immunohistochemistry using antibodies to Epstein-Barr Virus, CD21 and CD23 was done monthly. If abnormalities were noted, further immunological studies were done retrospectively. Immunofluorescence microscopy was undertaken in a different laboratory unaware of the patients underlying pathology. For this study, 6 patients diagnosed with episodes of mild rejection (,level 2), or none, were selected and results were compared with the two PTLD patients. A similar protocol was used for the cytoskeleton structural protein study using specific antibodies to microtubules and intermediate filaments.
Tissue Preparation Electron microscopy. Follow-up biopsies were processed for electron microscopy. Tissue samples were fixed in cacodylate buffer (0.2 mol/L, pH 7.4) containing 2% paraformaldehyde and 2.5% glutaraldehyde. After several washes in cacodylate buffer (0.1 mol/L) containing 11.25% of sucrose, biopsies were postfixed in 1% osmium tetroxide. Selected specimens were further treated with uranyl acetate “en bloc.” Biopsies were then dehydrated in a series of graded ethanol baths, incubated in propylene oxide and embedded in Epon 812.6 Semi-thin sections (1 mm thick) stained with 1% toluidine blue in a solution of 1% borax/50% ethanol, were routinely prepared in parallel for light microscopy to verify orientation of the tissue fibers. Ultra thin sections (400 to 600 Å) were stained in lead citrate and examined with a Jeol JEM-100CX II electron microscope. Light microscopy. Biopsies prepared for light microscopy were fixed and embedded in glycol methacrylate (GMA)7 and/or dehydrated through a graded series of ethanol baths and embedded in paraffin or polyethylene glycol (PEG).8,9 Different embedding media were necessary because of problems encountered with fixative used and antigen accessibility for each of these techniques. Tissue sections (3 to 4 mm thick) were cut with a Leitz microtome, mounted on glass slides and then stained with hematoxylin-eosin and Masson’s Trichrome stain. Immunohistochemistry and immunofluorescence. Slides embedded in paraffin were deparaffinated, treated with 0.3% hydrogen peroxide and rehydrated to distilled water. GMA and PEG sections were simply soaked in PBS to remove the embedding medium. Following rehydration in PBS buffer, slides were preincubated in blocking solution (PBS-BSA 0.3%) and then incubated with primary monoclonal antibodies to viral elements. After several washes, slides were incubated with a secondary antibody conjugated with biotin; a fluorochrome coupled to streptavidin was used for immunofluorescence microscopy and/or the antigen–antibody complex was revealed by an enzymatic detection system using peroxidase-streptavidin labeling. Control experiments were performed by exposing tissue sections to secondary antibody alone. When possible, immunohistochemical and immunofluorescence evaluation were done by two investigators using either staining technique in parallel. List of antibodies: 1. Epstein-Barr virus (Monoclonal EBNA, clone 0211, Biodesign International, Kennebunk, Me). 2. CD21 (Monoclonal anti-CD21, clone BU32, Biodesign International, Kennebunk Me; EBV receptor). 3. CD23 (Monoclonal anti-CD23, clone 9P25, Biodesign International, Kennebunk, Me; B-cell activation molecule).
919 4. Monoclonal antibody to HLA-DR, clone CR3/43, DaKopatts A/S, Denmark, was used to detect activated T lymphocytes. To evaluate the integrity of selected cytoskeletal structural proteins, the following antibodies were used: 1. Microtubules: a monoclonal anti-atubulin antibody, (clone DM1A, Sigma Immunochemical, St Louis, Md) 2. Intermediate filaments: a polyclonal anti-desmin antibody (kindly furnished by Doctor Michel Vincent, Laval University, Qc).
MORPHOLOGICAL RESULTS Rejection Study
Biopsy samples from the two PTLD patients indicated the occurrence of several episodes of severe rejection. Numerous activated T-lymphocytes were observed using HLA-DR labeling and were accompanied by cellular damage to cardiomyocytes. Occasional HLA-DR staining was observed in small foci in biopsies from patients with mild rejection but there was no apparent cellular damage to cardiomyocytes. Viral study
Immunofluorescence detection of EBV in cardiac biopsies four weeks before severe rejection showed intense labeling within damaged myocytes and in myocytes surrounding the damaged area (Fig 1). Peroxidase immunostaining was similarly distributed. EBV label was localized along the sarcolemma, within the nucleus and within lymphocyte cytoplasm surrounding myocytes. Two to 3 weeks before these observations, EBV peroxidase staining was observed in cardiac myocyte sarcolemma before any indication of staining within the nuclei. Viral nuclear proteins were identified in the subsarcolemmal region in a string of beads-like pattern. These bead-like structures may represent the formation of superantigens (Fig 2). This observation was paralleled by a fourfold increase in EBV titers in the blood. Electron microscopic analysis of endomyocardial biopsies from the two PTLD patients showed viral inclusions within the nucleus of cardiac myocytes (Fig 3). These inclusions consisted of a dense core surrounded by a clear halo; structures were closely related to the nucleolar material. EBV Receptor (CD21) and B-Cell Activation Molecule (CD23)
Immunolocalisation of CD21 using peroxidase and immunofluorescence techniques showed a focal positive staining at the cardiac myocyte sarcolemma. CD21 receptors in longitudinal sections of cardiac biopsies revealed a similar periodicity as EBV distribution (Fig 4). Positive staining of CD23 in myocytes was noted eight weeks before development of severe graft rejection and before development of PTLD. Longitudinal sections of endomyocardial biopsies also showed CD23 peroxidase staining at the sarcolemma (Fig 5); immunofluorescence microscopy confirmed these results. Staining of EBV, CD21 and CD23 was not detected
920
LEBLANC, BOUDRIAU, DOYLE ET AL
Fig 1. Immunofluorescence of cardiac myocytes showing strong reaction for EBV (EBNA) within numerous damaged myocytes and in myocytes surrounding the damaged area (PEG section). (Original magnification 3400).
in other selected patients with the exception of the two PTLD patients.
CYTOSKELETON STUDY
Immunofluorescence localization of the microtubule network in endomyocardial biopsies without rejection from eight selected patients including the PTLD patients showed uniform distribution of filaments throughout the myocytes, predominantly in longitudinal orientations and around the nucleus. In cardiac biopsies from patients with mild rejection, an inhomogeneous decrease in intensity of fluores-
Fig 2. (A) Peroxidase staining of EBV in GMA section shows a definite pattern at the sarcolemma of several myocytes (Arrows). Original magnification 3125. (B) EBV labeling along the sarcolemmal membrane demonstrates a string of beadslike organization (Arrow). Original magnification 3400. (C) Note, at high magnification the periodicity and the peculiar distribution of EBV labeling within myocyte membrane (Arrows). Original magnification 31000.
cence labeling was observed occasionally in several myocytes; with severe rejection, most myocytes were completely devoid of microtubule labeling (Fig 6). Immunofluorescence labeling of intermediate filaments using a rabbit polyclonal antibody to desmin showed a periodic staining localized at the Z-lines of cardiac myocyte sarcomeres and at the intercalated discs in cardiac biopsies without rejection. Inhomogeneous reduction in intensity of fluorescence labelling was observed during mild rejection while complete loss of Z-line and intercalated disc staining occurred in myocytes from biopsies with severe rejection (Fig 7). Phase contrast optic showed damage to myofibrils
EPSTEIN-BARR VIRUS MEDIATED GRAFT REJECTION
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Fig 3. Electromicrograph of a cardiac myocyte infected by EBV. Note the vacuolar aspect of cardiocyte cytoplasm and the irregular shape of the nucleus. Original magnification 32700. Insert shows a higher magnification of viral particles (arrow). Original magnification 310,000.
Fig 4. Peroxidase staining of CD21 in GMA longitudinal section of endocardial biopsy shows a periodic sarcolemmal immunoreactivity similar to EBV in some myocytes (Arrows). Original magnification 31200.
Fig 5. Peroxidase staining of CD23 in GMA longitudinal section shows an intense immunoreactivity along the sarcolemmal membrane of cardiocytes (Arrow). Original magnification 3650.
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Fig 6. Immunofluorescence labeling of a-tubulin (PEG sections) in control myocytes (A) shows that microtubules are distributed throughout the myocytes predominantly in longitudinal orientation. In figure (B), note the marked and inhomogeneous decrease in the intensity of fluorescence labeling in EBV infected cardiac myocytes. Original magnification 3500.
and loss of lateral registry, in myocytes with reduced immunofluorescence staining. Finally, it is noteworthy that all patients were able to serve as their own control since biopsies with no rejection, with mild rejection and severe rejection in the same patient were studied; when possible, tissue sections were studied simultaneously on the same slide. The parallel results for peroxidase and immunofluorescence labeling techniques with different fixatives and embedding techniques reinforce the value of our findings. Although, peroxidase staining was also performed for the cytoskeletal study, immunofluorescence labeling gave a better resolution.
LEBLANC, BOUDRIAU, DOYLE ET AL
Fig 7. Immunofluorescence labeling of desmin (PEG sections) in control cells (A) shows localization of desmin filaments at Z-lines of cardiac sarcomeres and at intercalated discs. In figure (B) EBV infected cells show an inhomogeneous decrease in the intensity of fluorescence labeling and a complete loss of Z-line and intercalated disc staining (lower right side). Original magnification 3500.
DISCUSSION
The findings reported here show that patients with severe cardiac rejection and extranodal lymphoma (PTLD) had viral inclusions in cardiac myocyte nuclei weeks before clinical manifestations of severe rejection and lymphoma. A positive localization of EBV in the cardiac myocyte sarcolemma, cytoplasm and nucleus was demonstrated in postgraft endomyocardial biopsies using a monoclonal EBV antibody (peroxidase and fluorescence). Focal detection of EBV receptor (CD21) was observed along the sarcolemma
EPSTEIN-BARR VIRUS MEDIATED GRAFT REJECTION
similar to EBV location. B-cell activation molecule (CD23) also strongly labeled cardiac myocyte sarcolemma. An extensive review of the current literature indicated that these findings have not previously been reported in cardiac tissue. These new findings are important since they may represent viral superantigens in association with MHC class II molecules evidenced by the periodicity of EBV at the cardiac myocyte subsarcolemma. The concept of viral superantigens may partly explain the development of severe rejection in cardiac transplantation since viral superantigens are known to recruit large numbers of lymphocytes (ie, up to 10,000 times).2 There is some evidence for superantigen formation in human tissues.10 In this study, positive immunostaining of HLA-DR (activated T-lymphocytes) was also observed in the area of infected myocytes (data not shown). At present, there is no evidence for EBV receptors (CD21) in normal cardiac myocytes under normal physiological conditions; this also holds true for skeletal muscle cells.11 In this study, CD21 was not found in tissue biopsies from selected patients in the absence of cardiac rejection. Biopsies from patients with mild rejection did not reveal CD21 on myocytes sarcolemma. Positive CD21 immunostaining paralleled the appearance and location of EBV at the level of cardiac myocyte sarcolemma. Immunoperoxidase labeling of EBV nuclear protein showed large cytoplasmic molecular complexes which were localized at periodic intervals along the sarcolemma. Thus, under pathological conditions, CD21 is present in cardiac myocytes. This data indicates that CD21 may help EBV binding to cardiac myocytes. A similar phenomenon has been reported earlier in skeletal muscle from patients with immunodeficiency syndrome (AIDS); EBV receptor CD21 was shown to be present in skeletal muscle cell tumors.11 High level of EBV was also present.11–13 The demonstration of CD23 in cardiac myocyte sarcolemma (a cluster domain known to stimulate B-cell proliferation), is unique. The fact that this molecule is localized on the cardiac myocyte sarcolemma weeks prior to clinical manifestation of extranodal lymphoma is of clinical importance. Early detection of CD23 may constitute a prelymphoma marker and as such, a warning for lymphoma development. This would allow for early detection of PTLD and hopefully a better clinical outcome. Several studies have emphasized a role for CD23 in relation to EBV associated B-cell lymphoma EBV.14 –16 These findings raise several interesting questions. Are CD21 and CD23 synthesized by cardiac myocytes? Are they translocated from lymphocyte membranes or are they transferred from interstitial fluid by endocytosis? Further studies are needed to clarify these questions. Finally, we show that cytoskeletal structural proteins including a-tubulin (microtubules) and desmin (intermediate filaments) are disorganized and reduced in EBV infected biopsies compared to normal cardiac myocytes.
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These results may provide an explanation for cardiac contractile dysfunction, which occurs in severe graft rejection. Morphological observations indicate that the association of EBV with the cardiac sarcolemma leads to rupture of cytoskeletal links between the sarcolemma and myofibrils. This finding is supported by several studies, both in vitro and in vivo, which document that viral infection in various cell types result in disruption of microtubules and the intermediate filament network and thereby provokes severe dysfunction in these cells.3–17 However, it is worth noting that CSK abnormalities may also occur in cardiomyocytes without viral infection as described in several patients with mild rejection. Furthermore, there is evidence that CSK disturbance in ischemic and hypertrophic heart is closely related to myocyte contractile dysfunction.18 –19 In summary, we show that EBV infection modulates severe graft rejection and precedes extranodal lymphoma in this group of cardiac transplant patients. EBV receptor (CD21) was observed at cardiac myocyte sarcolemma in association with B-cell activating factor CD23. EBV infection can produce important structural alterations at the level of microtubule and intermediate filament network which may lead to cardiac myocyte contractile dysfunction. These findings should shed some light on the pathogenesis of two important complications of cardiac transplantation, ie, severe rejection and extranodal B-cell lymphoma; these complications result in serious clinical morbidity and occasionally in mortality. ACKNOWLEDGMENTS We would like to thank Mrs Miche`le Be´langer, R.T., for technical assistance and Mrs Danielle Goulet for secretarial work. This work has been presented at the International Symposium “Current Problems in Heart Failure and Cardiac Transplantation: Assessment of Acute Rejection and Allograft Coronary Disease.” Thun, Switzerland, July 11 to 12, 1997.
REFERENCES 1. Thomas JA, Crawford DH: In Rose ML, Yacoub MH (eds): London: Edward Arnold; 1993, p 243 2. Lotteau V: Me´decine Sci 9:772, 1993 3. Girard M, Hirth L, Lebeurier G, et al: Virologie Mole´culaire. Paris: Doin; 1989, p 474 4. Billingham ME, Cary NRB, Hammond ME, et al: Heart Rejection Study Group 9:103, 1990 5. Crawford DH, Edwards JMB: In Zukerman AJ, Banatvale JE, Pattison JE (eds): Principles and Practice of Clinical Virology. London: John Wiley and Sons Ltd; 1990; p 103 6. Turcotte H, Bazin M, Boutet M: J Ultrastructure Res 72:133, 1982 7. Britten K, Roche WR: Clin Exp Allergy 22:123A, 1992 8. Boudriau S, Vincent M, Co ˆte´ CH, et al: J Histochem Cytochem 4:1013, 1993 9. Thibodeau A, Duhaime J, Simard J, et al: Histochem J 21:348, 1989 10. Lafon M, Lepage M, Martinez-Arends A, et al: Nature 358:507, 1992
924 11. McClain KL, Leach CT, Jenson HB, et al: New Engl J Med 332:12, 1995 12. Lee ES, Locker J, Nalesnik M, et al: New Engl J Med 332:19, 1995 13. Liebowitz DL: New Engl J Med 332:55, 1995 14. Randawa PS, Yousem SA, Paradis IL, et al: Am J Clinical Pathol 92:177, 1989
LEBLANC, BOUDRIAU, DOYLE ET AL 15. Weiss L, Movahed LA: Am J Pathol 134:651, 1989 16. Hanto DW, Sakamoto K, Purtilo DT, et al: Surgery 90:204, 1981 17. Zauli D, Musiani M, Crespi C, et al: Pathology 20:105, 1988 18. Sage MD, Jennings RB: Am J Pathol 133:327, 1988 19. Tsutsui H, Ishihara K, Cooper G IV: Science 260:682, 1993
D
URING the last decade, cardiac transplantation has evolved from experimental procedure to a selected choice for patients suffering from end-stage heart failure. Myocardial ischemia and dilated cardiomyopathy are the most common diseases requiring heart transplantation. However, the success of transplantation is often undermined by serious complications such as graft rejection, infection and neoplasia. Because recipients are treated with high doses of immunosuppressive medication, malignancy develops. One of the most common lymphomas in transplant patients is B-cell associated post-transplant lymphoproliferative disorder (PTLD). Up to 12% of recipients surviving more than 1 month after transplant develop PTLD and, in most cases, there is evidence that EpsteinBarr virus (EBV) may accompany or precede development of the disease.1 In contrast, EBV may be involved in graft rejection by a mechanism involving superantigens. The association of virus with class II major histocompatibility complex (MHC), may induce the formation of superantigens.2 These superantigens are presented to T-lymphocytes in their native form without previous degradation into small peptides. Superantigenic stimulation recruits up to 105-fold more T-lymphocytes compared to conventional antigens, which may induce severe rejection.2 Moreover, when viruses invade cells, they are known to disturb various cellular processes including synthesis and protein assembly.3 In this study, we hypothesized that EBV infecting cardiac myocytes may disrupt cytoskeleton structural proteins, which attach myofibrils to the sarcolemma; this could lead to cardiac contractile dysfunction. The principal objectives were to demonstrate the presence of EBV in cardiac myocytes from heart transplant patients diagnosed with severe graft rejection and extranodal lymphoma and to search for the EBV receptor CD21 and for B-cell activating factor CD23. In a parallel study, we examined the integrity of the cardiac myocyte cytoskeleton in viral infected endomyocardial biopsies to assess the potential relation between viral infection and cardiac myocyte cytoskeleton disruption. MATERIALS AND METHODS Heart Transplant Recipients Twenty-six patients underwent cardiac transplantation between 1993 and 1996. They were all treated with induction therapy 0041-1345/98/$19.00 PII S0041-1345(98)00096-7 918
comprising rabbit anti-thymocyte globulin (RATG) 125 mg (IV) for 3 days followed by standard triple regimen therapy (prednisone, cyclosporine, and azathioprine). Biopsy procedures were performed as part of the routine endomyocardial biopsy (EMB) protocol to monitor patients for acute rejection (weekly for 6 weeks, bimonthly for 2 months and monthly for 6 months). When severe rejection occurred, biopsies were sampled more frequently. EMB were obtained from the right interventricular septum with a bioptome introduced through the right internal jugular vein. Five to six pieces of endomyocardial tissue were obtained during each procedure; one specimen was used for electron microscopy; the others were prepared at random for light microscopy techniques (routine histology, immunohistology and immunofluorescence). Rejection was histologically diagnosed in EMB according to The International Society for Heart and Lung Transplantation (ISHLT) criteria.4 Two patients underwent several episodes of severe rejection and heart failure and developed extranodal lymphoma (PTLD); they died 3 to 6 months after the diagnosis of PTLD. The first patient had a primary lung tumor with secondary involvement of the liver and spleen. The second patient had a primary cardiac tumor at the level of the suture line between the donor and recipient pulmonary artery. Serological tests for cytomegalovirus (CMV) and EBV viral capsid antibody-IgG (VCA-IgG) were done in all these patients5; they were all EBV positive before transplantation; 14 were CMV positive.
Test CMV serological test. This test was performed on whole blood. White blood cells were extracted and concentrated by cytospin. They were stained by immunofluorescence with a monoclonal CMV antibody (Argene-Biosoft, Varilhes, Fr) and positive cells were numbered on a total of 2 3 105 cells. Results were expressed in percentage. EBV serological test. Four different antibodies were detected by titration and immunofluorescence. Tests were considered positive when VCA-IgG and VCA-IgM reached a concentration of 1/100, EBNA 1/10, and EA 1/80 (Caltag Laboratories Inc, San Francisco, Calif).
From the Quebec Heart Institute and Department of Pathology, Laval University, Ste-Foy, Quebec, Canada. Address reprint requests to Dr Marie-H. LeBlanc, Quebec Heart Institute, 2725, Chemin Ste-Foy, Ste-Foy, (Quebec), G1V 4G5, Canada. © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 30, 918–924 (1998)
EPSTEIN-BARR VIRUS MEDIATED GRAFT REJECTION
Rationale Electron microscopy and routine histology were performed after each biopsy procedure in all patients studied. Immunohistochemistry using antibodies to Epstein-Barr Virus, CD21 and CD23 was done monthly. If abnormalities were noted, further immunological studies were done retrospectively. Immunofluorescence microscopy was undertaken in a different laboratory unaware of the patients underlying pathology. For this study, 6 patients diagnosed with episodes of mild rejection (,level 2), or none, were selected and results were compared with the two PTLD patients. A similar protocol was used for the cytoskeleton structural protein study using specific antibodies to microtubules and intermediate filaments.
Tissue Preparation Electron microscopy. Follow-up biopsies were processed for electron microscopy. Tissue samples were fixed in cacodylate buffer (0.2 mol/L, pH 7.4) containing 2% paraformaldehyde and 2.5% glutaraldehyde. After several washes in cacodylate buffer (0.1 mol/L) containing 11.25% of sucrose, biopsies were postfixed in 1% osmium tetroxide. Selected specimens were further treated with uranyl acetate “en bloc.” Biopsies were then dehydrated in a series of graded ethanol baths, incubated in propylene oxide and embedded in Epon 812.6 Semi-thin sections (1 mm thick) stained with 1% toluidine blue in a solution of 1% borax/50% ethanol, were routinely prepared in parallel for light microscopy to verify orientation of the tissue fibers. Ultra thin sections (400 to 600 Å) were stained in lead citrate and examined with a Jeol JEM-100CX II electron microscope. Light microscopy. Biopsies prepared for light microscopy were fixed and embedded in glycol methacrylate (GMA)7 and/or dehydrated through a graded series of ethanol baths and embedded in paraffin or polyethylene glycol (PEG).8,9 Different embedding media were necessary because of problems encountered with fixative used and antigen accessibility for each of these techniques. Tissue sections (3 to 4 mm thick) were cut with a Leitz microtome, mounted on glass slides and then stained with hematoxylin-eosin and Masson’s Trichrome stain. Immunohistochemistry and immunofluorescence. Slides embedded in paraffin were deparaffinated, treated with 0.3% hydrogen peroxide and rehydrated to distilled water. GMA and PEG sections were simply soaked in PBS to remove the embedding medium. Following rehydration in PBS buffer, slides were preincubated in blocking solution (PBS-BSA 0.3%) and then incubated with primary monoclonal antibodies to viral elements. After several washes, slides were incubated with a secondary antibody conjugated with biotin; a fluorochrome coupled to streptavidin was used for immunofluorescence microscopy and/or the antigen–antibody complex was revealed by an enzymatic detection system using peroxidase-streptavidin labeling. Control experiments were performed by exposing tissue sections to secondary antibody alone. When possible, immunohistochemical and immunofluorescence evaluation were done by two investigators using either staining technique in parallel. List of antibodies: 1. Epstein-Barr virus (Monoclonal EBNA, clone 0211, Biodesign International, Kennebunk, Me). 2. CD21 (Monoclonal anti-CD21, clone BU32, Biodesign International, Kennebunk Me; EBV receptor). 3. CD23 (Monoclonal anti-CD23, clone 9P25, Biodesign International, Kennebunk, Me; B-cell activation molecule).
919 4. Monoclonal antibody to HLA-DR, clone CR3/43, DaKopatts A/S, Denmark, was used to detect activated T lymphocytes. To evaluate the integrity of selected cytoskeletal structural proteins, the following antibodies were used: 1. Microtubules: a monoclonal anti-atubulin antibody, (clone DM1A, Sigma Immunochemical, St Louis, Md) 2. Intermediate filaments: a polyclonal anti-desmin antibody (kindly furnished by Doctor Michel Vincent, Laval University, Qc).
MORPHOLOGICAL RESULTS Rejection Study
Biopsy samples from the two PTLD patients indicated the occurrence of several episodes of severe rejection. Numerous activated T-lymphocytes were observed using HLA-DR labeling and were accompanied by cellular damage to cardiomyocytes. Occasional HLA-DR staining was observed in small foci in biopsies from patients with mild rejection but there was no apparent cellular damage to cardiomyocytes. Viral study
Immunofluorescence detection of EBV in cardiac biopsies four weeks before severe rejection showed intense labeling within damaged myocytes and in myocytes surrounding the damaged area (Fig 1). Peroxidase immunostaining was similarly distributed. EBV label was localized along the sarcolemma, within the nucleus and within lymphocyte cytoplasm surrounding myocytes. Two to 3 weeks before these observations, EBV peroxidase staining was observed in cardiac myocyte sarcolemma before any indication of staining within the nuclei. Viral nuclear proteins were identified in the subsarcolemmal region in a string of beads-like pattern. These bead-like structures may represent the formation of superantigens (Fig 2). This observation was paralleled by a fourfold increase in EBV titers in the blood. Electron microscopic analysis of endomyocardial biopsies from the two PTLD patients showed viral inclusions within the nucleus of cardiac myocytes (Fig 3). These inclusions consisted of a dense core surrounded by a clear halo; structures were closely related to the nucleolar material. EBV Receptor (CD21) and B-Cell Activation Molecule (CD23)
Immunolocalisation of CD21 using peroxidase and immunofluorescence techniques showed a focal positive staining at the cardiac myocyte sarcolemma. CD21 receptors in longitudinal sections of cardiac biopsies revealed a similar periodicity as EBV distribution (Fig 4). Positive staining of CD23 in myocytes was noted eight weeks before development of severe graft rejection and before development of PTLD. Longitudinal sections of endomyocardial biopsies also showed CD23 peroxidase staining at the sarcolemma (Fig 5); immunofluorescence microscopy confirmed these results. Staining of EBV, CD21 and CD23 was not detected
920
LEBLANC, BOUDRIAU, DOYLE ET AL
Fig 1. Immunofluorescence of cardiac myocytes showing strong reaction for EBV (EBNA) within numerous damaged myocytes and in myocytes surrounding the damaged area (PEG section). (Original magnification 3400).
in other selected patients with the exception of the two PTLD patients.
CYTOSKELETON STUDY
Immunofluorescence localization of the microtubule network in endomyocardial biopsies without rejection from eight selected patients including the PTLD patients showed uniform distribution of filaments throughout the myocytes, predominantly in longitudinal orientations and around the nucleus. In cardiac biopsies from patients with mild rejection, an inhomogeneous decrease in intensity of fluores-
Fig 2. (A) Peroxidase staining of EBV in GMA section shows a definite pattern at the sarcolemma of several myocytes (Arrows). Original magnification 3125. (B) EBV labeling along the sarcolemmal membrane demonstrates a string of beadslike organization (Arrow). Original magnification 3400. (C) Note, at high magnification the periodicity and the peculiar distribution of EBV labeling within myocyte membrane (Arrows). Original magnification 31000.
cence labeling was observed occasionally in several myocytes; with severe rejection, most myocytes were completely devoid of microtubule labeling (Fig 6). Immunofluorescence labeling of intermediate filaments using a rabbit polyclonal antibody to desmin showed a periodic staining localized at the Z-lines of cardiac myocyte sarcomeres and at the intercalated discs in cardiac biopsies without rejection. Inhomogeneous reduction in intensity of fluorescence labelling was observed during mild rejection while complete loss of Z-line and intercalated disc staining occurred in myocytes from biopsies with severe rejection (Fig 7). Phase contrast optic showed damage to myofibrils
EPSTEIN-BARR VIRUS MEDIATED GRAFT REJECTION
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Fig 3. Electromicrograph of a cardiac myocyte infected by EBV. Note the vacuolar aspect of cardiocyte cytoplasm and the irregular shape of the nucleus. Original magnification 32700. Insert shows a higher magnification of viral particles (arrow). Original magnification 310,000.
Fig 4. Peroxidase staining of CD21 in GMA longitudinal section of endocardial biopsy shows a periodic sarcolemmal immunoreactivity similar to EBV in some myocytes (Arrows). Original magnification 31200.
Fig 5. Peroxidase staining of CD23 in GMA longitudinal section shows an intense immunoreactivity along the sarcolemmal membrane of cardiocytes (Arrow). Original magnification 3650.
922
Fig 6. Immunofluorescence labeling of a-tubulin (PEG sections) in control myocytes (A) shows that microtubules are distributed throughout the myocytes predominantly in longitudinal orientation. In figure (B), note the marked and inhomogeneous decrease in the intensity of fluorescence labeling in EBV infected cardiac myocytes. Original magnification 3500.
and loss of lateral registry, in myocytes with reduced immunofluorescence staining. Finally, it is noteworthy that all patients were able to serve as their own control since biopsies with no rejection, with mild rejection and severe rejection in the same patient were studied; when possible, tissue sections were studied simultaneously on the same slide. The parallel results for peroxidase and immunofluorescence labeling techniques with different fixatives and embedding techniques reinforce the value of our findings. Although, peroxidase staining was also performed for the cytoskeletal study, immunofluorescence labeling gave a better resolution.
LEBLANC, BOUDRIAU, DOYLE ET AL
Fig 7. Immunofluorescence labeling of desmin (PEG sections) in control cells (A) shows localization of desmin filaments at Z-lines of cardiac sarcomeres and at intercalated discs. In figure (B) EBV infected cells show an inhomogeneous decrease in the intensity of fluorescence labeling and a complete loss of Z-line and intercalated disc staining (lower right side). Original magnification 3500.
DISCUSSION
The findings reported here show that patients with severe cardiac rejection and extranodal lymphoma (PTLD) had viral inclusions in cardiac myocyte nuclei weeks before clinical manifestations of severe rejection and lymphoma. A positive localization of EBV in the cardiac myocyte sarcolemma, cytoplasm and nucleus was demonstrated in postgraft endomyocardial biopsies using a monoclonal EBV antibody (peroxidase and fluorescence). Focal detection of EBV receptor (CD21) was observed along the sarcolemma
EPSTEIN-BARR VIRUS MEDIATED GRAFT REJECTION
similar to EBV location. B-cell activation molecule (CD23) also strongly labeled cardiac myocyte sarcolemma. An extensive review of the current literature indicated that these findings have not previously been reported in cardiac tissue. These new findings are important since they may represent viral superantigens in association with MHC class II molecules evidenced by the periodicity of EBV at the cardiac myocyte subsarcolemma. The concept of viral superantigens may partly explain the development of severe rejection in cardiac transplantation since viral superantigens are known to recruit large numbers of lymphocytes (ie, up to 10,000 times).2 There is some evidence for superantigen formation in human tissues.10 In this study, positive immunostaining of HLA-DR (activated T-lymphocytes) was also observed in the area of infected myocytes (data not shown). At present, there is no evidence for EBV receptors (CD21) in normal cardiac myocytes under normal physiological conditions; this also holds true for skeletal muscle cells.11 In this study, CD21 was not found in tissue biopsies from selected patients in the absence of cardiac rejection. Biopsies from patients with mild rejection did not reveal CD21 on myocytes sarcolemma. Positive CD21 immunostaining paralleled the appearance and location of EBV at the level of cardiac myocyte sarcolemma. Immunoperoxidase labeling of EBV nuclear protein showed large cytoplasmic molecular complexes which were localized at periodic intervals along the sarcolemma. Thus, under pathological conditions, CD21 is present in cardiac myocytes. This data indicates that CD21 may help EBV binding to cardiac myocytes. A similar phenomenon has been reported earlier in skeletal muscle from patients with immunodeficiency syndrome (AIDS); EBV receptor CD21 was shown to be present in skeletal muscle cell tumors.11 High level of EBV was also present.11–13 The demonstration of CD23 in cardiac myocyte sarcolemma (a cluster domain known to stimulate B-cell proliferation), is unique. The fact that this molecule is localized on the cardiac myocyte sarcolemma weeks prior to clinical manifestation of extranodal lymphoma is of clinical importance. Early detection of CD23 may constitute a prelymphoma marker and as such, a warning for lymphoma development. This would allow for early detection of PTLD and hopefully a better clinical outcome. Several studies have emphasized a role for CD23 in relation to EBV associated B-cell lymphoma EBV.14 –16 These findings raise several interesting questions. Are CD21 and CD23 synthesized by cardiac myocytes? Are they translocated from lymphocyte membranes or are they transferred from interstitial fluid by endocytosis? Further studies are needed to clarify these questions. Finally, we show that cytoskeletal structural proteins including a-tubulin (microtubules) and desmin (intermediate filaments) are disorganized and reduced in EBV infected biopsies compared to normal cardiac myocytes.
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These results may provide an explanation for cardiac contractile dysfunction, which occurs in severe graft rejection. Morphological observations indicate that the association of EBV with the cardiac sarcolemma leads to rupture of cytoskeletal links between the sarcolemma and myofibrils. This finding is supported by several studies, both in vitro and in vivo, which document that viral infection in various cell types result in disruption of microtubules and the intermediate filament network and thereby provokes severe dysfunction in these cells.3–17 However, it is worth noting that CSK abnormalities may also occur in cardiomyocytes without viral infection as described in several patients with mild rejection. Furthermore, there is evidence that CSK disturbance in ischemic and hypertrophic heart is closely related to myocyte contractile dysfunction.18 –19 In summary, we show that EBV infection modulates severe graft rejection and precedes extranodal lymphoma in this group of cardiac transplant patients. EBV receptor (CD21) was observed at cardiac myocyte sarcolemma in association with B-cell activating factor CD23. EBV infection can produce important structural alterations at the level of microtubule and intermediate filament network which may lead to cardiac myocyte contractile dysfunction. These findings should shed some light on the pathogenesis of two important complications of cardiac transplantation, ie, severe rejection and extranodal B-cell lymphoma; these complications result in serious clinical morbidity and occasionally in mortality. ACKNOWLEDGMENTS We would like to thank Mrs Miche`le Be´langer, R.T., for technical assistance and Mrs Danielle Goulet for secretarial work. This work has been presented at the International Symposium “Current Problems in Heart Failure and Cardiac Transplantation: Assessment of Acute Rejection and Allograft Coronary Disease.” Thun, Switzerland, July 11 to 12, 1997.
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