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Nuclear Mean Gray Level and Chromatin Distribution Changes in Cardiomyocytes of Heart Transplant Recipients Suffering From Acute Cellular Rejection
Nuclear Mean Gray Level and Chromatin Distribution Changes in Cardiomyocytes of Heart Transplant Recipients Suffering From Acute Cellular Rejection M. Zakliczynski, J. Nozynski, D. Lange, E. Zembala-Nozynska, D. Konecka-Mrówka, and M. Zembala ABSTRACT Background. Distribution and staining of the nuclear chromatin are sensitive indicators of changes in physiology and pathology of cells. However, their use in heart transplant recipients is rare. The aim of this study was to compare cardiomyocyte status in heart transplant recipients suffering from moderate acute cellular rejection and subjects without signs of active cellular rejection. Materials and Methods. One hundred twenty-nine endomyocardial biopsy samples from 43 heart transplant recipients (no later than 6 months after surgery) were evaluated. Overall, 3235 cardiomyocytic nuclei were analyzed using the Quantimet image analysis system to assess the mean gray level: 1584 nuclei were found in biopsies without signs of rejection or with nonsignificant rejection (ISHLT grades 0, 1A, and 1B), whereas the remaining 1651 nuclei were measured from biopsies showing ISHLT grade 3A (significant rejection). Additionally, the chromatin distribution was assessed in all eligible nuclei. Results. The mean gray level was markedly increased in nuclei from biopsy samples with significant rejection (182.6 vs 112.5, P ⬍ .001, Mann-Whitney U test). Moreover, the analysis of chromatin distribution revealed significantly more frequent chromatin marginalization and irregular distribution in rejecting subjects (P ⬍ .001, chi-square test). Conclusion. Inflammatory stimulation of cardiomyocytes during acute cellular rejection of the transplanted heart influences the chromatin distribution in the nuclei, which may have an additional value, when assessing the severity of rejection.
D
ISTRIBUTION AND STAINING of the nuclear chromatin are thought to be sensitive indicators of changes in physiology, mitotic status, and pathology of cells. It is known that chromatin becomes bright and delicately granular or irregularly despiralized in all stimulated cells, whereas in resting or nonstimulating cells it is dark and clumped. In the first descriptions of cardiac allograft pathology, only during acute moderate cellular rejection was the cardiocytic chromatin morphology mentioned.1–5 Thereafter attention was turned to inflammatory infiltrates, and cardiomyocytic nuclear morphology was thought to be less important. In fact, inflammatory infiltrations represent a main but not the sole sign of rejection, whereas cardiocytic nuclear morphology reflects the cardiocytic stimulation, regenerative, and/or adaptive answer for local cellular, humoral, and molecular pathological events during rejection. However, evaluation of cardiocytic chromatin in heart transplant rejection has not been studied yet. The aim of
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 38, 325–327 (2006)
this study was to compare cardiomyocyte status in heart transplant recipients suffering from moderate acute cellular rejection and subjects without signs of active cellular rejection. MATERIALS AND METHODS A total of 129 endomyocardial biopsy (EMB) samples from 43 heart transplant recipients (no later than 6 months after surgery) From the Department of Cardiac Surgery and Transplantation, Silesian Center for Heart Diseases (M.Za., J.N., D.K.-M., M.Ze.), Zabrze, Poland; Department of Pathology, Institute of Oncology (D.L.), Gliwice, Poland; and Department of Pathology, Silesian Medical University (E.Z.-N.), Zabrze, Poland. Address reprint requests to Michal Zakliczynski, Department of Cardiac Surgery and Transplantation, Silesian Center for Heart Diseases, ul. Szpitalna 2, 41-800 Zabrze, Poland. E-mail: zaklimed@ onet.pl 0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2005.12.029 325
326
Fig 1.
ZAKLICZYNSKI, NOZYNSKI, LANGE ET AL
Mean gray levels of cardiocytic nuclear chromatin.
were evaluated. The control group consisted of 10 heart valve donors, between 23 and 28 years old, without any cardiovascular pathology. In all cases, the EMBs were fixed in nonaqueous fluid (Carnoy fixative), sparing nuclear RNA and glycogen, and shortening the diagnostic procedure. Then the tissue was proceeded routinely to paraffin. The slices (5 m thick) were stained with standardized Harris hematoxylin for 5 minutes, then washed in tap water (10 minutes), counterstained with eosin, and after dehydration, covered with DPX. The Quantimet Color Option image analysis system was used, and under 1000-fold magnification the cardiocytic nuclei were marked using tablet. Overall, 3235 cardiomyocytic nuclei were analyzed to assess the mean gray level: 1584 nuclei were found in biopsies without signs of rejection or with nonsignificant rejection (nonaggressive rejection group, ISHLT grades 0, 1A, and 1B), whereas the remaining 1651 nuclei were measured from biopsies showing ISHLT grade 3A (aggressive, or significant rejection). The control group was represented by 2000 measurements. Additionally, the chromatin distribution was assessed in all eligible nuclei. The Mann-Whitney U test and chi-square test were used for statistical analyses.
RESULTS
The mean gray levels of nuclear chromatin are shown in Fig. 1. The mean gray levels in the control group and not aggressively rejecting group were comparable with no significant difference (control, 120.6 ⫾ 51.3; no aggressive
Fig 2. Chromatin distribution: comparisons of regular and irregular distribution.
rejection, 112.5 ⫾ 42.3; P ⬍ .05). The control and nonrejecting groups differed significantly with aggressively rejecting biopsies (aggressive rejection, 182.6 ⫾ 61.3), and the differences were highly significant. The chromatin distributions in the analyzed groups are given in Figs. 2 and 3. In the control and nonrejecting myocardial samples the chromatin was predominantly regularly distributed, but in nonrejecting EMBs the percentage of that distribution was significantly smaller than in the control group. The difference becomes highly significant when comparing the control with the aggressively rejecting group or the nonrejecting with the aggressively rejecting group. The comparisons of marginated and central/pericentral patterns showed a lack of differences between the control and aggressively rejecting groups differed significantly. DISCUSSION
Attempts to use image analysis in the microscopic cardiac transplant assessing procedure have indicated mainly signs of cardiomyocytic nuclei enlargement, suggesting hypertrophy.6 From many suspected agents, interleukin-2 (IL-2) tumor necrosis factor-alpha, and ventricular adrenomedullin were suspected as the main agents, involved in long-term hypertrophic response.7–9 These changes should be responsible for the ventricular wall remodeling, without any signs of cardiac failure, as in senescent or overloaded or injured hearts.10 –12 The cardiocytic nuclear changes during the rejection episode were not extensively commented on in the available literature; moreover, we have not found any studies in which a major constituent of cardiomyocytic nuclei, nuclear chromatin, was evaluated in the same individuals during acute aggressive rejection (grade 3A), and when they were free from rejection. Our former morphologic studies of cardiomyocytic chromatin have indicated that it reacts by changing its condensation, reflecting in normo- or hyperchromasia under various therapeutic protocols.13 The changes in chromatin stainability reflect its decondensation, chromatin remodeling.14,15 This recent
CARDIOMYOCYTES IN ACUTE CELLULAR REJECTION
327
Fig 3. Chromatin distribution: comparisons of marginated and central/pericentral patterns.
study with the use of the image analysis system indicates that cardiocytic chromatin reflects immunological disturbances in interstitial space during rejection as an influence of immunosuppressive drugs. The mean gray level, an objective parameter of chromatin condensation, was not significantly higher in the control group compared with the nonaggressive rejection group. It may reflect an inhibitory effect of a standard immunosuppressive protocol, not for cardiomyocytic hypertrophic response but for chromatin stimulation. On the other hand, the irregular chromatin distribution was significantly more frequent in the nonrejecting group, suggesting spiralization. The group with the aggressive rejection (3A) presented significantly brighter, irregular, and marginated chromatin, ie, despiralized and active. These results indicate that lymphocytes are not the only effector cells during rejection activation which present signs of stimulation. Chromatin remodeling takes place also in cardiomyocytes, probably under the influence of IL2,14 –16 or IL-4/14,17 but not immunosuppressants, also administered in the comparative group. Moreover, the assessment of the chromatin distribution, stainability, or mean gray level should be helpful as an indicator of cardiocytic stimulation in EMBs. In conclusion, inflammatory stimulation of cardiomyocytes during acute cellular rejection of the transplanted heart influences the chromatin distribution in the nuclei. These cytologic changes may have an additional value when assessing the severity of cellular rejection. REFERENCES 1. Bieber CP, Stinson EB, Shumway NE, et al: Cardiac transplantation in man. VII. Cardiac allograft pathology. Circulation 41:753, 1970 2. Caves PK, Billingham ME, Schulz WP, et al: Transvenous biopsy from canine orthotopic heart allografts. Am Heart J 85:525, 1973 3. Caves PK, Stinson EB, Billingham ME, et al: Diagnosis of human cardiac allograft rejection by serial cardiac biopsy. J Thorac Cardiovasc Surg 461:66, 1973
4. Caves PK, Stinson EC, Billingham ME, et al: Serial transvenous biopsy of the transplanted human heart: improved management of acute rejection episodes. Lancet 1(7862):821, 1974 5. Billingham ME: Diagnosis of cardiac rejection by endomyocardial biopsy. Heart Transplant 25:1, 1981 6. Rowan RA, Billingham ME: Sustained myocardial hypertrophy seven years or more after heart transplantation: a morphometric study of endomyocardial specimens. J Heart Lung Transplant 11:350, 1992 7. Herrera Garza EH, Herrera Garza JL, Rodriguez Gonzalez H, et al: Importance of tumor necrosis factor-alpha in the pathogenesis of heart failure. Rev Esp Cardiol 55:61, 2002 8. Stetson SJ, Perez-Verdia A, Mazur W, et al: Cardiac hypertrophy after transplantation is associated with persistent expression of tumor necrosis factor-alpha. Circulation 104:676, 2001 9. Tsuruda T, Jougasaki M, Boerrigter G, et al: Ventricular adrenomedullin is associated with myocyte hypertrophy in human transplanted heart. Regul Pep 112:161, 2003 10. Olivetti G, Melissari M, Balbi T, et al: Myocyte nuclear and possible cellular hyperplasia contribute to ventricular remodeling in the hypertrophic senescent heart in humans. J Am Coll Cardiol 24:140, 1994 11. Olivetti G, Melissari M, Balbi T, et al: Myocyte cellular hypertrophy is responsible for ventricular remodeling in the hypertrophied heart of middle-aged individuals in the absence of cardiac failure. Cardiovasc Res 28:1199, 1994 12. Olivetti G, Quaini F, Lagrasta C, et al: Myocyte cellular hypertrophy and hyperplasia contribute to ventricular wall remodeling in anemia-induced cardiac hypertrophy in rats. Am J Pathol 141:227, 1992 13. Zakliczynski M, Nozynski J, Swierad M, et al: Pathologic assessment of cardiomyocytes in heart transplant recipients treated with rapamycine or cyclosporine. Transplant Proc 35:2329, 2003 14. Rao S, Procko E, Shannon MF: Chromatin remodeling, measured by a novel real-time polymerase chain reaction assay, across the proximal promoter region of the IL-2 gene. J Immunol 167:4494, 2001 15. Smale ST, Fisher AG: Chromatin structure and gene regulation in the immune system. Annu Rev Immunol 20:427, 2002 16. Ward SB, Hernandez-Hoyos G, Chen F, et al: Chromatin remodeling of the interleukin-2 gene: distinct alterations in the proximal versus distal enhancer regions. Nucleic Acids Res 26: 2923, 1998 17. Takemoto N, Kamogawa Y, Jun Lee H, et al: Cutting edge: chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific cytokine gene cluster. J Immunol 165:6687, 2000
D
ISTRIBUTION AND STAINING of the nuclear chromatin are thought to be sensitive indicators of changes in physiology, mitotic status, and pathology of cells. It is known that chromatin becomes bright and delicately granular or irregularly despiralized in all stimulated cells, whereas in resting or nonstimulating cells it is dark and clumped. In the first descriptions of cardiac allograft pathology, only during acute moderate cellular rejection was the cardiocytic chromatin morphology mentioned.1–5 Thereafter attention was turned to inflammatory infiltrates, and cardiomyocytic nuclear morphology was thought to be less important. In fact, inflammatory infiltrations represent a main but not the sole sign of rejection, whereas cardiocytic nuclear morphology reflects the cardiocytic stimulation, regenerative, and/or adaptive answer for local cellular, humoral, and molecular pathological events during rejection. However, evaluation of cardiocytic chromatin in heart transplant rejection has not been studied yet. The aim of
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 38, 325–327 (2006)
this study was to compare cardiomyocyte status in heart transplant recipients suffering from moderate acute cellular rejection and subjects without signs of active cellular rejection. MATERIALS AND METHODS A total of 129 endomyocardial biopsy (EMB) samples from 43 heart transplant recipients (no later than 6 months after surgery) From the Department of Cardiac Surgery and Transplantation, Silesian Center for Heart Diseases (M.Za., J.N., D.K.-M., M.Ze.), Zabrze, Poland; Department of Pathology, Institute of Oncology (D.L.), Gliwice, Poland; and Department of Pathology, Silesian Medical University (E.Z.-N.), Zabrze, Poland. Address reprint requests to Michal Zakliczynski, Department of Cardiac Surgery and Transplantation, Silesian Center for Heart Diseases, ul. Szpitalna 2, 41-800 Zabrze, Poland. E-mail: zaklimed@ onet.pl 0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2005.12.029 325
326
Fig 1.
ZAKLICZYNSKI, NOZYNSKI, LANGE ET AL
Mean gray levels of cardiocytic nuclear chromatin.
were evaluated. The control group consisted of 10 heart valve donors, between 23 and 28 years old, without any cardiovascular pathology. In all cases, the EMBs were fixed in nonaqueous fluid (Carnoy fixative), sparing nuclear RNA and glycogen, and shortening the diagnostic procedure. Then the tissue was proceeded routinely to paraffin. The slices (5 m thick) were stained with standardized Harris hematoxylin for 5 minutes, then washed in tap water (10 minutes), counterstained with eosin, and after dehydration, covered with DPX. The Quantimet Color Option image analysis system was used, and under 1000-fold magnification the cardiocytic nuclei were marked using tablet. Overall, 3235 cardiomyocytic nuclei were analyzed to assess the mean gray level: 1584 nuclei were found in biopsies without signs of rejection or with nonsignificant rejection (nonaggressive rejection group, ISHLT grades 0, 1A, and 1B), whereas the remaining 1651 nuclei were measured from biopsies showing ISHLT grade 3A (aggressive, or significant rejection). The control group was represented by 2000 measurements. Additionally, the chromatin distribution was assessed in all eligible nuclei. The Mann-Whitney U test and chi-square test were used for statistical analyses.
RESULTS
The mean gray levels of nuclear chromatin are shown in Fig. 1. The mean gray levels in the control group and not aggressively rejecting group were comparable with no significant difference (control, 120.6 ⫾ 51.3; no aggressive
Fig 2. Chromatin distribution: comparisons of regular and irregular distribution.
rejection, 112.5 ⫾ 42.3; P ⬍ .05). The control and nonrejecting groups differed significantly with aggressively rejecting biopsies (aggressive rejection, 182.6 ⫾ 61.3), and the differences were highly significant. The chromatin distributions in the analyzed groups are given in Figs. 2 and 3. In the control and nonrejecting myocardial samples the chromatin was predominantly regularly distributed, but in nonrejecting EMBs the percentage of that distribution was significantly smaller than in the control group. The difference becomes highly significant when comparing the control with the aggressively rejecting group or the nonrejecting with the aggressively rejecting group. The comparisons of marginated and central/pericentral patterns showed a lack of differences between the control and aggressively rejecting groups differed significantly. DISCUSSION
Attempts to use image analysis in the microscopic cardiac transplant assessing procedure have indicated mainly signs of cardiomyocytic nuclei enlargement, suggesting hypertrophy.6 From many suspected agents, interleukin-2 (IL-2) tumor necrosis factor-alpha, and ventricular adrenomedullin were suspected as the main agents, involved in long-term hypertrophic response.7–9 These changes should be responsible for the ventricular wall remodeling, without any signs of cardiac failure, as in senescent or overloaded or injured hearts.10 –12 The cardiocytic nuclear changes during the rejection episode were not extensively commented on in the available literature; moreover, we have not found any studies in which a major constituent of cardiomyocytic nuclei, nuclear chromatin, was evaluated in the same individuals during acute aggressive rejection (grade 3A), and when they were free from rejection. Our former morphologic studies of cardiomyocytic chromatin have indicated that it reacts by changing its condensation, reflecting in normo- or hyperchromasia under various therapeutic protocols.13 The changes in chromatin stainability reflect its decondensation, chromatin remodeling.14,15 This recent
CARDIOMYOCYTES IN ACUTE CELLULAR REJECTION
327
Fig 3. Chromatin distribution: comparisons of marginated and central/pericentral patterns.
study with the use of the image analysis system indicates that cardiocytic chromatin reflects immunological disturbances in interstitial space during rejection as an influence of immunosuppressive drugs. The mean gray level, an objective parameter of chromatin condensation, was not significantly higher in the control group compared with the nonaggressive rejection group. It may reflect an inhibitory effect of a standard immunosuppressive protocol, not for cardiomyocytic hypertrophic response but for chromatin stimulation. On the other hand, the irregular chromatin distribution was significantly more frequent in the nonrejecting group, suggesting spiralization. The group with the aggressive rejection (3A) presented significantly brighter, irregular, and marginated chromatin, ie, despiralized and active. These results indicate that lymphocytes are not the only effector cells during rejection activation which present signs of stimulation. Chromatin remodeling takes place also in cardiomyocytes, probably under the influence of IL2,14 –16 or IL-4/14,17 but not immunosuppressants, also administered in the comparative group. Moreover, the assessment of the chromatin distribution, stainability, or mean gray level should be helpful as an indicator of cardiocytic stimulation in EMBs. In conclusion, inflammatory stimulation of cardiomyocytes during acute cellular rejection of the transplanted heart influences the chromatin distribution in the nuclei. These cytologic changes may have an additional value when assessing the severity of cellular rejection. REFERENCES 1. Bieber CP, Stinson EB, Shumway NE, et al: Cardiac transplantation in man. VII. Cardiac allograft pathology. Circulation 41:753, 1970 2. Caves PK, Billingham ME, Schulz WP, et al: Transvenous biopsy from canine orthotopic heart allografts. Am Heart J 85:525, 1973 3. Caves PK, Stinson EB, Billingham ME, et al: Diagnosis of human cardiac allograft rejection by serial cardiac biopsy. J Thorac Cardiovasc Surg 461:66, 1973
4. Caves PK, Stinson EC, Billingham ME, et al: Serial transvenous biopsy of the transplanted human heart: improved management of acute rejection episodes. Lancet 1(7862):821, 1974 5. Billingham ME: Diagnosis of cardiac rejection by endomyocardial biopsy. Heart Transplant 25:1, 1981 6. Rowan RA, Billingham ME: Sustained myocardial hypertrophy seven years or more after heart transplantation: a morphometric study of endomyocardial specimens. J Heart Lung Transplant 11:350, 1992 7. Herrera Garza EH, Herrera Garza JL, Rodriguez Gonzalez H, et al: Importance of tumor necrosis factor-alpha in the pathogenesis of heart failure. Rev Esp Cardiol 55:61, 2002 8. Stetson SJ, Perez-Verdia A, Mazur W, et al: Cardiac hypertrophy after transplantation is associated with persistent expression of tumor necrosis factor-alpha. Circulation 104:676, 2001 9. Tsuruda T, Jougasaki M, Boerrigter G, et al: Ventricular adrenomedullin is associated with myocyte hypertrophy in human transplanted heart. Regul Pep 112:161, 2003 10. Olivetti G, Melissari M, Balbi T, et al: Myocyte nuclear and possible cellular hyperplasia contribute to ventricular remodeling in the hypertrophic senescent heart in humans. J Am Coll Cardiol 24:140, 1994 11. Olivetti G, Melissari M, Balbi T, et al: Myocyte cellular hypertrophy is responsible for ventricular remodeling in the hypertrophied heart of middle-aged individuals in the absence of cardiac failure. Cardiovasc Res 28:1199, 1994 12. Olivetti G, Quaini F, Lagrasta C, et al: Myocyte cellular hypertrophy and hyperplasia contribute to ventricular wall remodeling in anemia-induced cardiac hypertrophy in rats. Am J Pathol 141:227, 1992 13. Zakliczynski M, Nozynski J, Swierad M, et al: Pathologic assessment of cardiomyocytes in heart transplant recipients treated with rapamycine or cyclosporine. Transplant Proc 35:2329, 2003 14. Rao S, Procko E, Shannon MF: Chromatin remodeling, measured by a novel real-time polymerase chain reaction assay, across the proximal promoter region of the IL-2 gene. J Immunol 167:4494, 2001 15. Smale ST, Fisher AG: Chromatin structure and gene regulation in the immune system. Annu Rev Immunol 20:427, 2002 16. Ward SB, Hernandez-Hoyos G, Chen F, et al: Chromatin remodeling of the interleukin-2 gene: distinct alterations in the proximal versus distal enhancer regions. Nucleic Acids Res 26: 2923, 1998 17. Takemoto N, Kamogawa Y, Jun Lee H, et al: Cutting edge: chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific cytokine gene cluster. J Immunol 165:6687, 2000