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Acute vascular humoral rejection in a sensitized cardiac graft recipient: diagnostic value of C4d immunofluorescence
CASE STUDIES
ACUTE VASCULAR HUMORAL REJECTION IN A SENSITIZED CARDIAC GRAFT RECIPIENT: DIAGNOSTIC VALUE OF C4D IMMUNOFLUORESCENCE JEAN-PAUL DUONG VAN HUYEN, MD, PAUL FORNES, MD, PHD, ROMAIN GUILLEMAIN, MD, CATHERINE AMREIN, MD, PATRICK CHEVALIER, MD, CHRISTIAN LATREMOUILLE, MD, PHD, CAROLINE CREPUT, MD, DENIS GLOTZ, MD, PHD, DOMINIQUE NOCHY, MD, AND PATRICK BRUNEVAL, MD A 37-year-old female patient had a cardiac transplantation for dilated cardiomyopathy. She was sensitized by two pregnancies showing anti– human leukocyte antigen I and II antibodies. The pretransplantation crossmatch was negative, but she developed acute humoral rejection characterized by vascular C4d deposits, arteriolitis, and intravascular leukocyte accumulation and adhesion in venules. Although C4d deposits disappeared in 4 weeks, she had persistent endothelial cell activation (endothelial expression of ELAM-1, VCAM-1, or human leukocyte antigen class II) throughout the 6 months of follow-up. Although she received intensive immunosuppression, she presented three episodes of acute cellular rejection
during that period of time. This case shows that C4d deposits represent a sensitive marker of acute humoral rejection in cardiac transplantation. Therefore, C4d immunofluorescence should be more frequently assessed in endomyocardial biopsies. HUM PATHOL 35: 385-388. © 2004 Elsevier Inc. All rights reserved. Key words: cardiac transplantation, humoral rejection, C4d, endomyocardial biopsy. Abbreviations: EMB, endomyocardial biopsies; HLA, human leukocyte antigen; ELAM-1, endothelial leucocyte adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1.
Although microvascular endothelial C4d deposits characterizing acute humoral rejection have been frequently reported in renal transplantation,1,2 they seem very rare in heart transplantation.3 Furthermore, the natural history and consequences of humoral rejection remain unclear and are the scope of most recent studies in cardiac transplantation.4 We report here such a case in which sequential endomyocardial biopsies allow new insights into the phenomenon of C4d-positive acute humoral rejection.
plantation tests showed patient sensitization: presence of antiHLA class I and II IgG by ELISA and panel reactive antibodies at 62% by microlymphocytotoxicity. No antiendothelial or antiepithelial cell specificities were detected. Several months before transplantation, the patient was treated with intravenous immune globulin (IVIg; Gammagard, Baxter, Maurepas, France) regimens that did not reverse the sensitization status. The HLA groups were A2, A24; B8, B60; DR7, DR15 for the recipient and A31, 66; B35, 41; DR4, DR7 for the donor. In this sensitized patient, the prospective donor-specific crossmatch performed before the transplantation was negative. The recipient had the standard immunosuppression at our institution: induction with anti-human thymocyte Ig (Thymoglobulin, Sangstat, Lyon, France), together with mycofenolate mofetil (3 g/d, through mycophenolic acid [MPA] at 3.98 ng/mL), cyclosporine (200 ng/mL), and prednisone (1 mg/ kg/d). The posttransplantation clinical and echocardiographic monitoring remained unremarkable for the 6 months of follow-up. At day 7, the first EMB showed an unusual pattern of rejection, including dilatation of venules with blood leukocyte accumulation (Fig 1A) and leukocyte adhesion to the endothelium, rare arteriolitis without fibrin necrosis (Fig 1B), and intense C4d deposits in all the interstitial capillaries, a small artery, and the endocardium at immunofluorescence on frozen sections (Fig 1C and D). C3, C1q, and fibrin (Fig 1E) were positive in some capillaries. IgG was negative and IgM weakly positive in most capillaries. A few arteriolar endothelial cells were activated as shown by strong expression of HLA class II and VCAM-1. Most of the intravascular mononuclear cells were CD68-positive macrophages (Fig 1F) as well as rare interstitial cells, whereas CD3-positive T lymphocytes were absent. Given that pattern of acute humoral vascular rejection, the patient received IV methylprednisone (1 g for 3 days), and tacrolimus (targeted to a through level of 15 ng/mL) instead of cyclosporine, and IVIg (80 g for 3 days). Intense C4d capillary deposits persisted up to day 28 (4th EMB) and remained constantly negative thereafter, up to 6 months. However, markers of endothelial cell activation (VCAM-1, ELAM-1, and HLA class II expression) increased from the second EMB, spreading to most of the interstitial capillaries and persisting up to 6 months (Fig 1G and H). At day 10, ELISA showed the appearance of donor-specific antibodies (IgG anti-A66). At day 21 (third EMB), the first cellular acute rejection episode was diagnosed as a grade 3A of ISHLT classification. Its cellular composition was unusual, the CD68-positive macrophages being prominent over the CD3-
MATERIALS AND METHODS Right ventricle endomyocardial biopsies (EMB) were snap-frozen (1 specimen) or fixed in formalin (4 to 6 specimens). Immunohistochemistry was performed on cryostat sections for C4d (Quidel, Santa Clara, CA), C3, C1q, fibrin, IgG, IgM, HLA class II (all from Dako, Trappes, France), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leucocyte adhesion molecule-1 (ELAM-1) (both from R&D Systems Europe, Abington, UK) and on paraffin sections for CD1a, CD3, CD 20, CD68, and CD79a (all from Dako). Acute cellular rejection diagnosis was based on the analysis of 8 hematoxylin and eosin–stained sections of all the biopsied specimens according to the International Society for Heart and Lung Transplantation (ISHLT) grading system.5 As controls, we assessed the first EMB in the 20 last transplanted patients (not sensitized) in our institution with C4d immunofluorescence: no C4d deposits were found. CASE REPORT In November 2002, a 37-year-old female patient underwent orthotopic cardiac transplantation for peripartum dilated cardiomyopathy that had evolved for 18 months. She had 4 children, 2 twins from 2 pregnancies. The cardiomyopathy started in the 9th month of the last pregnancy. Pretrans-
From the Department of Pathology, Unit of Cardiac Transplantation, and Department of Nephrology, Hoˆ pital Europe´ en Georges Pompidou, Paris, France. Accepted for publication October 15, 2003. Address correspondence and reprint requests to Patrick Bruneval, MD, Department of Pathology, Hoˆ pital Europe´ en Georges Pompidou, 20 rue Leblanc, 75015-Paris, France. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/S0046-8177(03)00621-X
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CASE STUDIES
TABLE 1. Follow-Up on Sequential Endomyocardial Biopsies Parameter
Data
Biopsy number (d) 1 (7) 2 (14) 3 (21) 4 (28) 5 (42) 6 (58) 7 (75) 8 (94) 9 (120) 10 (145) 11 (175) Acute cellular rejection grade in ISHLT grading system 0 arteriolitis 0 3A 1B arteriolitis 0 0 1B 1A 3A 0 1A Fluorescence deposits and endothelial activation markersa C4d ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ Fibrin, C3, C1q ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ IgG ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ IgM ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ HLA-DR ⫾ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ELAM-I ⫾ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ VCAM-I ⫾ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ Cell lineage markersb CD68 (localization) ⫹⫹ (IV) ⫹⫹ (IV) ⫹⫹⫹ (IT) ⫹⫹ (IT) ⫺ ⫺ ⫹⫹ (IT) ⫺ ⫹ (IT) ⫺ ⫾ (IT) CD3 (localization) ⫺ ⫺ ⫹ (IT) ⫹⫹ (IT) ⫺ ⫺ ⫹ (IT) ⫹ ⫹⫹⫹ (IT) ⫺ ⫹ (IT) CD20, CD79a, CD1a ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ Abbreviations: ISHLT: International Society for Heart and Lung Transplantation grading system for acute cellular rejection5; IT, intratissular; IV, intravascular leukocytes. a ⫺, negative; ⫾, weakly positive; ⫹, positive. b ⫺, no cell; ⫾, rare cells; from ⫹ to ⫹⫹⫹, some to many cells.
positive T lymphocytes. It was successfully treated with thymoglobulin and IV methylprednisone (1 g for 3 days). At day 75 (seventh EMB), a 1B-grade acute rejection episode was detected (the CD68-positive macrophages were still prominent over the CD3-positive T lymphocytes) and treated with OKT3 (Cilag) and IV methylprednisone (1 g for 3 days). It was completely resolved on the eighth EMB. But at day 120 (ninth EMB), a 3A-grade acute rejection episode was diagnosed, showing a classical pattern of prominent CD3-positive T lymphocytes over rare CD68-positive macrophages. An IV methylprednisone (1 g for 3 days) pulse was given, and sirolimus (targeted to a through level of 12 ng/mL) was added to the treatment. At days 145 and 175 (10th and 11th EMBs), no more significant acute cellular rejection was detected, but endothelial cell activation persisted. See Table 1 for a summary of the follow-up results. DISCUSSION This is a case of acute humoral vascular rejection in a sensitized recipient who developed an anti-HLA class I response against the cardiac graft. It is remarkable for initial and transient C4d deposits in the graft vasculature, together with persistent endothelial cell activation, intravascular macrophages, and arteriolitis. Therefore, C4d deposits are clearly a component of the pathological features characterizing acute humoral rejection. They appear to be a sensitive marker of acute humoral rejection in cardiac transplantation, as is the case in renal transplantation. Acute humoral rejection is rarely reported in cardiac transplantation,3,4 contrary to the case in renal transplanta-
tion, where C4d deposits are now considered to be the gold standard for the diagnosis of renal graft acute humoral rejection.1,2 In addition, C4d deposits have also been associated with a component of humoral rejection in the chronic allograft nephropathy.6 The diagnosis of cardiac acute humoral rejection is lacking accuracy on EMB given the facts that C4d immunofluorescence has not been performed as extensively as in renal transplantation and that there has been controversy about the value of immunoglobulin and complement deposits on EMB to detect humoral rejection.7,8 Furthermore, C4d deposits have been described in early ischemic graft injury without any initial immune mechanism.9 Therefore, the diagnosis of acute humoral rejection on EMB and especially the use of C4d immunofluorescence need further studies to be supported. However, in our case the C4d deposits are likely linked to acute humoral rejection because they are observed in a sensitized recipient who developed donorspecific antibodies and they are associated with arteriolitis and C3, fibrin, and C1q deposits. The EMB showed an unusual pattern of leukocyte intravascular accumulation and adhesion in the microcirculation with initial prominent macrophage recruitment. Such a pattern has been rarely described,4,10 but it could be a marker of humoral rejection and endothelial cell activation. This should be systematically assessed on EMB analysis as well as C4d immunofluorescence to improve the diagnostic value of EMB to detect humoral rejection. Cardiac humoral rejection has been associated with inconstant adverse events.3,4 In our case, during the first 6 months of follow-up, no clinical or echographic changes were
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ FIGURE 1. (A) Intravascular leukocyte accumulation in EMB 1. (H&E; ⫻800.) (B) Arteriolitis without fibrin necrosis in EMB 1. (H&E; ⫻600.) (C) Numerous C4d deposits in the endocardium and the microvessels on EMB 2. (Immunofluorescence; ⫻20.) (D) C4d deposits in the vascular endothelial cells of numerous interstitial capillaries on EMB 2. (Immunofluorescence; ⫻800.) (E) Fibrin deposits in some interstitial capillaries on EMB 4. (Immunofluorescence; ⫻400.) (F) Intravascular CD68 monocytes accumulated within a venule and adherent to the endothelium on EMB 2. (CD68 immunohistochemistry; ⫻800.) (G) Strong expression of human leukocyte antigen class II in many endothelial cells reflecting endothelial cell activation on EMB 10. (Human leukocyte antigen class II immunohistochemistry; ⫻500.) (H) Expression of VCAM-1 in the endothelial cells of a venule on EMB 10. (VCAM-1 immunohistochemistry; ⫻1000.)
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observed. These observations support the fact that acute humoral rejection does not always have an early catastrophic effect but may be silent. However, the intensive immunosuppression given to our patient as early as day 7 (first EMB), when humoral rejection was detected, could be a factor lessening the expression of humoral rejection. Thus, cardiac humoral rejection can induce subtle EMB changes, of which the pathologist has now to be aware by systematically looking for C4d deposits, intravascular leukocyte accumulation, initial macrophage recruitment, and endothelial cell activation. Endothelial cells express no or little ELAM-1, VCAM-1, or HLA class II markers of endothelial cell activation in nonrejecting cardiac allografts, whereas they express these molecules in the classical pattern of acute cellular rejection.11-13 In our case, this expression preceded acute cellular rejection by several weeks and persisted unchanged when the acute cellular rejection was successfully treated. This suggests that donor-specific antibodies in this sensitized recipient could induce sustained endothelial cell activation, which in turn could be responsible for macrophage and T-lymphocyte recruitment and for recurrent episodes of acute cellular rejection. In conclusion, it is time now to assess EMB in a different manner, including new histological criteria and immunohistochemical markers, at least in a subset of transplanted patients, to detect humoral rejection, because this type of rejection factors in prognosis and treatment.14,15
REFERENCES 1. Feucht HE, Scheeberger H, Hillebrand G, et al: Capillary deposition of C4d complement fragment and early renal graft loss. Kidney Int 43:1333-1338, 1993 2. Nickeleit V, Zeiler M, Gudat F, et al: Detection of the complement degradation product C4d in renal allografts: Diagnostic and therapeutic implications. J Am Soc Nephrol 13:242-251, 2002
3. Behr TM, Feucht HE, Richter K, et al: Detection of humoral rejection in human cardiac allografts by assessing the capillary deposition of complement fragment C4d in endomyocardial biopsies. J Heart Lung Transplant 18:904-912, 1999 4. Michaels PJ, Espejo ML, Kobashigawa J, et al: Humoral rejection in cardiac transplantation: Risk factors, hemodynamic consequences and relationship to transplant coronary heart disease. J Heart Lung Transplant 22:58-69, 2003 5. Billingham ME, Cary NR, Hammond ME, et al: A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart Rejection Study Group. The International Society for Heart Transplantation. J Heart Transplant 9:587-593, 1990 6. Regele H, Bo¨ hmig GA, Habicht A, et al: Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular and glomerular capillaries: A contribution of humoral immunity to chronic allograft rejection. J Am Soc Nephrol 13:23712380, 2002 7. Hammond EH, Yowell RL, Nunoda S, et al: Vascular (humoral) rejection in heart transplantation: Pathologic observations and clinical implications. J Heart Transplant 8:430-443, 1989 8. Bonnaud EN, Lewis NP, Masek MA, et al: Reliability and usefulness of immunofluorescence in heart transplantation. J Heart Lung Transplant 14:163171, 1995 9. Baldwin WM 3rd, Samaniego-Picota M, Kasper EK, et al: Complement deposition in early cardiac transplant biopsies is associated with ischemic injury and subsequent rejection episodes. Transplantation 27:894-900, 1999 10. Ratliff NB, McMahon JT: Activation of intravascular macrophages within myocardial small vessels is a feature of acute vascular rejection in human heart transplants. J Heart Lung Transplant 14:338-345, 1995 11. Taylor PM, Rose ML, Yacoub MH, et al: Induction of vascular adhesion molecules during rejection of human cardiac allografts. Transplantation 54:451-457, 1992 12. Carlos T, Gordon D, Fishbein D, et al: Vascular cell adhesion molecule-1 is induced on endothelium during acute rejection in human cardiac allografts. J Heart Lung Transplant 11:1103-1109, 1992 13. Ferran C, Peuchmaur M, Desruennes J, et al: Implications of de novo ELAM-1 and VCAM-1 expression in human cardiac allograft rejection. Transplantation 55:605-609, 1993 14. Arenda JM, Scornik JC, Normann SJ, et al: Anti-CD20 monoclonal antibody (rituximab) therapy for acute cardiac humoral rejection: A case report. Transplantation 27:907-910, 2002 15. Garrett HE, Groshart K, Duvall-Seaman D, et al: Treatment of humoral rejection with rituximab. Ann Thorac Surg 74:1240-1242, 2002
ERRATUM
In Takeshita et al: Clinicopathologic Differences Between 22 Cases of CD56-Negative and CD56-Positive Subcutaneous Panniculitis-Like Lymphoma in Japan, HUMAN PATHOLOGY 35:231-239, February 2004, Shuhei Imayama, MD, was inadvertently omitted from the author byline. He was a coauthor of the article and should be noted as such. The correct list of authors is: Morishige Takeshita, MD, Seiichi Okamura, MD, Yumi Oshiro, MD, Shuhei Imayama, MD, Sumika Okamoto, MD, Yasumas Matsuki, MD, Yutaka Nakashima, MD, Takashi Okamura, MD, Motoaki Shiratsuchi, MD, Toru Hayashi, MD, and Masahiro Kikuchi, MD.
388
ACUTE VASCULAR HUMORAL REJECTION IN A SENSITIZED CARDIAC GRAFT RECIPIENT: DIAGNOSTIC VALUE OF C4D IMMUNOFLUORESCENCE JEAN-PAUL DUONG VAN HUYEN, MD, PAUL FORNES, MD, PHD, ROMAIN GUILLEMAIN, MD, CATHERINE AMREIN, MD, PATRICK CHEVALIER, MD, CHRISTIAN LATREMOUILLE, MD, PHD, CAROLINE CREPUT, MD, DENIS GLOTZ, MD, PHD, DOMINIQUE NOCHY, MD, AND PATRICK BRUNEVAL, MD A 37-year-old female patient had a cardiac transplantation for dilated cardiomyopathy. She was sensitized by two pregnancies showing anti– human leukocyte antigen I and II antibodies. The pretransplantation crossmatch was negative, but she developed acute humoral rejection characterized by vascular C4d deposits, arteriolitis, and intravascular leukocyte accumulation and adhesion in venules. Although C4d deposits disappeared in 4 weeks, she had persistent endothelial cell activation (endothelial expression of ELAM-1, VCAM-1, or human leukocyte antigen class II) throughout the 6 months of follow-up. Although she received intensive immunosuppression, she presented three episodes of acute cellular rejection
during that period of time. This case shows that C4d deposits represent a sensitive marker of acute humoral rejection in cardiac transplantation. Therefore, C4d immunofluorescence should be more frequently assessed in endomyocardial biopsies. HUM PATHOL 35: 385-388. © 2004 Elsevier Inc. All rights reserved. Key words: cardiac transplantation, humoral rejection, C4d, endomyocardial biopsy. Abbreviations: EMB, endomyocardial biopsies; HLA, human leukocyte antigen; ELAM-1, endothelial leucocyte adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1.
Although microvascular endothelial C4d deposits characterizing acute humoral rejection have been frequently reported in renal transplantation,1,2 they seem very rare in heart transplantation.3 Furthermore, the natural history and consequences of humoral rejection remain unclear and are the scope of most recent studies in cardiac transplantation.4 We report here such a case in which sequential endomyocardial biopsies allow new insights into the phenomenon of C4d-positive acute humoral rejection.
plantation tests showed patient sensitization: presence of antiHLA class I and II IgG by ELISA and panel reactive antibodies at 62% by microlymphocytotoxicity. No antiendothelial or antiepithelial cell specificities were detected. Several months before transplantation, the patient was treated with intravenous immune globulin (IVIg; Gammagard, Baxter, Maurepas, France) regimens that did not reverse the sensitization status. The HLA groups were A2, A24; B8, B60; DR7, DR15 for the recipient and A31, 66; B35, 41; DR4, DR7 for the donor. In this sensitized patient, the prospective donor-specific crossmatch performed before the transplantation was negative. The recipient had the standard immunosuppression at our institution: induction with anti-human thymocyte Ig (Thymoglobulin, Sangstat, Lyon, France), together with mycofenolate mofetil (3 g/d, through mycophenolic acid [MPA] at 3.98 ng/mL), cyclosporine (200 ng/mL), and prednisone (1 mg/ kg/d). The posttransplantation clinical and echocardiographic monitoring remained unremarkable for the 6 months of follow-up. At day 7, the first EMB showed an unusual pattern of rejection, including dilatation of venules with blood leukocyte accumulation (Fig 1A) and leukocyte adhesion to the endothelium, rare arteriolitis without fibrin necrosis (Fig 1B), and intense C4d deposits in all the interstitial capillaries, a small artery, and the endocardium at immunofluorescence on frozen sections (Fig 1C and D). C3, C1q, and fibrin (Fig 1E) were positive in some capillaries. IgG was negative and IgM weakly positive in most capillaries. A few arteriolar endothelial cells were activated as shown by strong expression of HLA class II and VCAM-1. Most of the intravascular mononuclear cells were CD68-positive macrophages (Fig 1F) as well as rare interstitial cells, whereas CD3-positive T lymphocytes were absent. Given that pattern of acute humoral vascular rejection, the patient received IV methylprednisone (1 g for 3 days), and tacrolimus (targeted to a through level of 15 ng/mL) instead of cyclosporine, and IVIg (80 g for 3 days). Intense C4d capillary deposits persisted up to day 28 (4th EMB) and remained constantly negative thereafter, up to 6 months. However, markers of endothelial cell activation (VCAM-1, ELAM-1, and HLA class II expression) increased from the second EMB, spreading to most of the interstitial capillaries and persisting up to 6 months (Fig 1G and H). At day 10, ELISA showed the appearance of donor-specific antibodies (IgG anti-A66). At day 21 (third EMB), the first cellular acute rejection episode was diagnosed as a grade 3A of ISHLT classification. Its cellular composition was unusual, the CD68-positive macrophages being prominent over the CD3-
MATERIALS AND METHODS Right ventricle endomyocardial biopsies (EMB) were snap-frozen (1 specimen) or fixed in formalin (4 to 6 specimens). Immunohistochemistry was performed on cryostat sections for C4d (Quidel, Santa Clara, CA), C3, C1q, fibrin, IgG, IgM, HLA class II (all from Dako, Trappes, France), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leucocyte adhesion molecule-1 (ELAM-1) (both from R&D Systems Europe, Abington, UK) and on paraffin sections for CD1a, CD3, CD 20, CD68, and CD79a (all from Dako). Acute cellular rejection diagnosis was based on the analysis of 8 hematoxylin and eosin–stained sections of all the biopsied specimens according to the International Society for Heart and Lung Transplantation (ISHLT) grading system.5 As controls, we assessed the first EMB in the 20 last transplanted patients (not sensitized) in our institution with C4d immunofluorescence: no C4d deposits were found. CASE REPORT In November 2002, a 37-year-old female patient underwent orthotopic cardiac transplantation for peripartum dilated cardiomyopathy that had evolved for 18 months. She had 4 children, 2 twins from 2 pregnancies. The cardiomyopathy started in the 9th month of the last pregnancy. Pretrans-
From the Department of Pathology, Unit of Cardiac Transplantation, and Department of Nephrology, Hoˆ pital Europe´ en Georges Pompidou, Paris, France. Accepted for publication October 15, 2003. Address correspondence and reprint requests to Patrick Bruneval, MD, Department of Pathology, Hoˆ pital Europe´ en Georges Pompidou, 20 rue Leblanc, 75015-Paris, France. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/S0046-8177(03)00621-X
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CASE STUDIES
TABLE 1. Follow-Up on Sequential Endomyocardial Biopsies Parameter
Data
Biopsy number (d) 1 (7) 2 (14) 3 (21) 4 (28) 5 (42) 6 (58) 7 (75) 8 (94) 9 (120) 10 (145) 11 (175) Acute cellular rejection grade in ISHLT grading system 0 arteriolitis 0 3A 1B arteriolitis 0 0 1B 1A 3A 0 1A Fluorescence deposits and endothelial activation markersa C4d ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ Fibrin, C3, C1q ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ IgG ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ IgM ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ HLA-DR ⫾ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ELAM-I ⫾ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ VCAM-I ⫾ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ Cell lineage markersb CD68 (localization) ⫹⫹ (IV) ⫹⫹ (IV) ⫹⫹⫹ (IT) ⫹⫹ (IT) ⫺ ⫺ ⫹⫹ (IT) ⫺ ⫹ (IT) ⫺ ⫾ (IT) CD3 (localization) ⫺ ⫺ ⫹ (IT) ⫹⫹ (IT) ⫺ ⫺ ⫹ (IT) ⫹ ⫹⫹⫹ (IT) ⫺ ⫹ (IT) CD20, CD79a, CD1a ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ Abbreviations: ISHLT: International Society for Heart and Lung Transplantation grading system for acute cellular rejection5; IT, intratissular; IV, intravascular leukocytes. a ⫺, negative; ⫾, weakly positive; ⫹, positive. b ⫺, no cell; ⫾, rare cells; from ⫹ to ⫹⫹⫹, some to many cells.
positive T lymphocytes. It was successfully treated with thymoglobulin and IV methylprednisone (1 g for 3 days). At day 75 (seventh EMB), a 1B-grade acute rejection episode was detected (the CD68-positive macrophages were still prominent over the CD3-positive T lymphocytes) and treated with OKT3 (Cilag) and IV methylprednisone (1 g for 3 days). It was completely resolved on the eighth EMB. But at day 120 (ninth EMB), a 3A-grade acute rejection episode was diagnosed, showing a classical pattern of prominent CD3-positive T lymphocytes over rare CD68-positive macrophages. An IV methylprednisone (1 g for 3 days) pulse was given, and sirolimus (targeted to a through level of 12 ng/mL) was added to the treatment. At days 145 and 175 (10th and 11th EMBs), no more significant acute cellular rejection was detected, but endothelial cell activation persisted. See Table 1 for a summary of the follow-up results. DISCUSSION This is a case of acute humoral vascular rejection in a sensitized recipient who developed an anti-HLA class I response against the cardiac graft. It is remarkable for initial and transient C4d deposits in the graft vasculature, together with persistent endothelial cell activation, intravascular macrophages, and arteriolitis. Therefore, C4d deposits are clearly a component of the pathological features characterizing acute humoral rejection. They appear to be a sensitive marker of acute humoral rejection in cardiac transplantation, as is the case in renal transplantation. Acute humoral rejection is rarely reported in cardiac transplantation,3,4 contrary to the case in renal transplanta-
tion, where C4d deposits are now considered to be the gold standard for the diagnosis of renal graft acute humoral rejection.1,2 In addition, C4d deposits have also been associated with a component of humoral rejection in the chronic allograft nephropathy.6 The diagnosis of cardiac acute humoral rejection is lacking accuracy on EMB given the facts that C4d immunofluorescence has not been performed as extensively as in renal transplantation and that there has been controversy about the value of immunoglobulin and complement deposits on EMB to detect humoral rejection.7,8 Furthermore, C4d deposits have been described in early ischemic graft injury without any initial immune mechanism.9 Therefore, the diagnosis of acute humoral rejection on EMB and especially the use of C4d immunofluorescence need further studies to be supported. However, in our case the C4d deposits are likely linked to acute humoral rejection because they are observed in a sensitized recipient who developed donorspecific antibodies and they are associated with arteriolitis and C3, fibrin, and C1q deposits. The EMB showed an unusual pattern of leukocyte intravascular accumulation and adhesion in the microcirculation with initial prominent macrophage recruitment. Such a pattern has been rarely described,4,10 but it could be a marker of humoral rejection and endothelial cell activation. This should be systematically assessed on EMB analysis as well as C4d immunofluorescence to improve the diagnostic value of EMB to detect humoral rejection. Cardiac humoral rejection has been associated with inconstant adverse events.3,4 In our case, during the first 6 months of follow-up, no clinical or echographic changes were
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ FIGURE 1. (A) Intravascular leukocyte accumulation in EMB 1. (H&E; ⫻800.) (B) Arteriolitis without fibrin necrosis in EMB 1. (H&E; ⫻600.) (C) Numerous C4d deposits in the endocardium and the microvessels on EMB 2. (Immunofluorescence; ⫻20.) (D) C4d deposits in the vascular endothelial cells of numerous interstitial capillaries on EMB 2. (Immunofluorescence; ⫻800.) (E) Fibrin deposits in some interstitial capillaries on EMB 4. (Immunofluorescence; ⫻400.) (F) Intravascular CD68 monocytes accumulated within a venule and adherent to the endothelium on EMB 2. (CD68 immunohistochemistry; ⫻800.) (G) Strong expression of human leukocyte antigen class II in many endothelial cells reflecting endothelial cell activation on EMB 10. (Human leukocyte antigen class II immunohistochemistry; ⫻500.) (H) Expression of VCAM-1 in the endothelial cells of a venule on EMB 10. (VCAM-1 immunohistochemistry; ⫻1000.)
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HUMAN PATHOLOGY
Volume 35, No. 3 (Month 2004)
observed. These observations support the fact that acute humoral rejection does not always have an early catastrophic effect but may be silent. However, the intensive immunosuppression given to our patient as early as day 7 (first EMB), when humoral rejection was detected, could be a factor lessening the expression of humoral rejection. Thus, cardiac humoral rejection can induce subtle EMB changes, of which the pathologist has now to be aware by systematically looking for C4d deposits, intravascular leukocyte accumulation, initial macrophage recruitment, and endothelial cell activation. Endothelial cells express no or little ELAM-1, VCAM-1, or HLA class II markers of endothelial cell activation in nonrejecting cardiac allografts, whereas they express these molecules in the classical pattern of acute cellular rejection.11-13 In our case, this expression preceded acute cellular rejection by several weeks and persisted unchanged when the acute cellular rejection was successfully treated. This suggests that donor-specific antibodies in this sensitized recipient could induce sustained endothelial cell activation, which in turn could be responsible for macrophage and T-lymphocyte recruitment and for recurrent episodes of acute cellular rejection. In conclusion, it is time now to assess EMB in a different manner, including new histological criteria and immunohistochemical markers, at least in a subset of transplanted patients, to detect humoral rejection, because this type of rejection factors in prognosis and treatment.14,15
REFERENCES 1. Feucht HE, Scheeberger H, Hillebrand G, et al: Capillary deposition of C4d complement fragment and early renal graft loss. Kidney Int 43:1333-1338, 1993 2. Nickeleit V, Zeiler M, Gudat F, et al: Detection of the complement degradation product C4d in renal allografts: Diagnostic and therapeutic implications. J Am Soc Nephrol 13:242-251, 2002
3. Behr TM, Feucht HE, Richter K, et al: Detection of humoral rejection in human cardiac allografts by assessing the capillary deposition of complement fragment C4d in endomyocardial biopsies. J Heart Lung Transplant 18:904-912, 1999 4. Michaels PJ, Espejo ML, Kobashigawa J, et al: Humoral rejection in cardiac transplantation: Risk factors, hemodynamic consequences and relationship to transplant coronary heart disease. J Heart Lung Transplant 22:58-69, 2003 5. Billingham ME, Cary NR, Hammond ME, et al: A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart Rejection Study Group. The International Society for Heart Transplantation. J Heart Transplant 9:587-593, 1990 6. Regele H, Bo¨ hmig GA, Habicht A, et al: Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular and glomerular capillaries: A contribution of humoral immunity to chronic allograft rejection. J Am Soc Nephrol 13:23712380, 2002 7. Hammond EH, Yowell RL, Nunoda S, et al: Vascular (humoral) rejection in heart transplantation: Pathologic observations and clinical implications. J Heart Transplant 8:430-443, 1989 8. Bonnaud EN, Lewis NP, Masek MA, et al: Reliability and usefulness of immunofluorescence in heart transplantation. J Heart Lung Transplant 14:163171, 1995 9. Baldwin WM 3rd, Samaniego-Picota M, Kasper EK, et al: Complement deposition in early cardiac transplant biopsies is associated with ischemic injury and subsequent rejection episodes. Transplantation 27:894-900, 1999 10. Ratliff NB, McMahon JT: Activation of intravascular macrophages within myocardial small vessels is a feature of acute vascular rejection in human heart transplants. J Heart Lung Transplant 14:338-345, 1995 11. Taylor PM, Rose ML, Yacoub MH, et al: Induction of vascular adhesion molecules during rejection of human cardiac allografts. Transplantation 54:451-457, 1992 12. Carlos T, Gordon D, Fishbein D, et al: Vascular cell adhesion molecule-1 is induced on endothelium during acute rejection in human cardiac allografts. J Heart Lung Transplant 11:1103-1109, 1992 13. Ferran C, Peuchmaur M, Desruennes J, et al: Implications of de novo ELAM-1 and VCAM-1 expression in human cardiac allograft rejection. Transplantation 55:605-609, 1993 14. Arenda JM, Scornik JC, Normann SJ, et al: Anti-CD20 monoclonal antibody (rituximab) therapy for acute cardiac humoral rejection: A case report. Transplantation 27:907-910, 2002 15. Garrett HE, Groshart K, Duvall-Seaman D, et al: Treatment of humoral rejection with rituximab. Ann Thorac Surg 74:1240-1242, 2002
ERRATUM
In Takeshita et al: Clinicopathologic Differences Between 22 Cases of CD56-Negative and CD56-Positive Subcutaneous Panniculitis-Like Lymphoma in Japan, HUMAN PATHOLOGY 35:231-239, February 2004, Shuhei Imayama, MD, was inadvertently omitted from the author byline. He was a coauthor of the article and should be noted as such. The correct list of authors is: Morishige Takeshita, MD, Seiichi Okamura, MD, Yumi Oshiro, MD, Shuhei Imayama, MD, Sumika Okamoto, MD, Yasumas Matsuki, MD, Yutaka Nakashima, MD, Takashi Okamura, MD, Motoaki Shiratsuchi, MD, Toru Hayashi, MD, and Masahiro Kikuchi, MD.
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