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Ann Thorac Surg 2001;71:S379-S384
© 2001 The Society of Thoracic Surgeons

Basic research

Immunological aspects of fresh and cryopreserved aortic valve transplantation in rats

Frans B.S. Oei, MDa, Alexander P.A. Stegmann, PhDa, Leonard M.B. Vaessen, PhDb, Richard L. Marquet, PhDc, Willem Weimar, MD, PhDb, Ad J.J.C. Bogers, MD, PhDa

a Department of Cardio-Thoracic Surgery, Heart Valve Bank Rotterdam, Rotterdam, The Netherlands
b Department of Internal Medicine I, Heart Valve Bank Rotterdam, Rotterdam, The Netherlands
c Department of Experimental Surgery, University Hospital Rotterdam, Rotterdam, The Netherlands

Address reprint requests to Dr Oei, Department of Cardio-Thoracic Surgery, University Hospital Rotterdam, Room Bd158, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands
e-mail: oei{at}inw1.azr.nl

Presented at the VIII International Symposium on Cardiac Bioprostheses, Cancun, Mexico, Nov 3–5, 2000.


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. The influence of immune activation on valve allograft degeneration remains unclear. We studied the combined effect of major histocompatibility complex (MHC)-incompatibility and cryopreservation on valve performance, histomorphology, and tissue antigenicity in rats.

Methods. Fresh or cryopreserved allogeneic aortic valves from WAG (RT1u) rats were transplanted to DA (RT1a) recipients and syngenic transplants served as controls. After 7 or 21 days, valves were examined for competence and morphology. Immune reactivity of the recipient was measured by concanavalin A (conA) stimulation and analysis of donor-reactive Helper T-lymphocyte frequencies (HTLf) in peripheral blood and spleen.

Results. Syngenic grafts demonstrated normal competence and structure. Allografts lost their competence over time caused by destruction of the leaflets combined with cellular infiltration in the vascular wall. Cryopreservation induces early loss of competence and retrovalvular thrombosis. Cryopreserved allografts were also heavily infiltrated. ConA stimulation indices and HTLf were higher in allogeneic recipients compared to syngenic recipients (p < 0.03). Cryopreserved allografts elicited a lower immune response compared with fresh allografts (p < 0.03).

Conclusions. Aortic valve allografts are able to induce a donor-reactive immune response that is related to early graft destruction and incompetence. Cryopreservation appears to diminish but not eliminate the antigenicity of the allograft.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Theoretically, valve allograft failure may be caused by immunological factors induced by blood group (ABO) or human leukocyte antigens (HLA) mismatch. Previous in vitro studies already demonstrated the antigenicity of human valve allografts [1], later confirmed by clinical ex vivo studies [2]. The method of cryopreservation, introduced by O’Brien and colleagues [3], improves long-term graft performance by retaining the viability of valvular cells. Nevertheless, viable valvular cells could express HLA antigens, which in HLA mismatched condition could trigger an allogeneic response leading to graft rejection. Echocardiographic monitoring of the aortic valve allograft in the patient can be useful for monitoring the valve function. For diagnosis of graft rejection, characterized by cellular infiltration and tissue necrosis, continuous histopathologic analysis of the transplanted tissue is necessary. This is certainly not possible in human valve allograft follow-up. Several in vivo studies using inbred rat strains have brought evidence for immune-mediated structural damage in fresh valve allografts [4]. However, the influence of cryopreservation in this process remains unclear.

In this article we describe the functional, pathologic, and immunological changes of aortic valve transplantation, by using an end-to-side heterotopic implantation model in rats with strong histoincompatibility [5]. In addition, the effect of cryopreservation on valve immunogenicity and structure was studied.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Experimental design
This study includes four experimental groups consisting of fresh allogeneic (FA), cryopreserved allogeneic (CA), fresh syngeneic (FS), and cryopreserved syngeneic (CS) aortic valve transplantations. Male inbred DA (RT-1a/RyHSD) and WAG-Rij (RT-1u/RyHSD) rats, weighing 200 to 250 g (Harlan CPB, Horst, The Netherlands), were used as recipients or syngeneic donors and allogeneic donors, respectively. A modified intraabdominal implantation model was used [5]. Each group included 12 animals, sacrificed either at day 7 (n = 6) or day 21 (n = 6) followed by extraction of peripheral blood, spleen, and valve graft. After gross examination of the graft, valve competence was analyzed by retrograde injection of saline. Subsequently, standard histologic and immunohistochemical evaluations were performed on all explanted grafts. Immune reactivity was measured among peripheral blood mononuclear cells (PBMC) and spleen cells using concanavalin A (conA) stimulation and donor-reactive T-lymphocyte frequency (HTLf) analysis. Surgical failures including ischemic or neurologic complications, preliminary death, obliteration of the graft, and aneurysm of the anastomosis were excluded from the study. The experimental protocols were concordant with the Guidelines on the Protection of Experimental Animals by the Council of the European Community (1986) and approved by the Committee on Animal Research of Erasmus University of Rotterdam (Rotterdam, The Netherlands).

Aortic valve grafts were directly implanted or transported to the Heart Valve Bank Rotterdam after procurement for sterilization, cryopreservation, and storage (at least 4 weeks) according to standard protocol [2]. Before implantation, the cryopreserved valves were thawed in a 37°C water bath and gently flushed with heparinized saline (50 U/mL). Before implantation, all valve grafts were checked for competence by retrograde injection with saline.

Histologic analysis
After competence testing and macroscopic examination of the leaflet, sinus of Valsalva, and vascular wall, the explanted grafts were dissected longitudinally into three symmetric rectangular pieces for standard histologic and immunohistochemical staining. Valve pieces were fixed in 10% buffered formaldehyde for at least 24 hours and embedded in paraffin. Hematoxylin-eosin stained 4 µm sections were examined under microscope by two independent investigators (F.B.S.O., A.P.A.S.). The presence of mononuclear cells infiltration in all three vascular wall layers and valve leaflet was scored semiquantitatively (grade 0 = normal, 1 = mild, 2 = moderate, and 3 = severe). For phenotyping the infiltrates, immunohistochemical staining of frozen valve sections (5 µm) was performed by a three-layer immunoperoxidase technique [5]. We used 5 different mouse-antirat IgG monoclonal primary antibodies (Serotec Europe, Oxford, UK) directed against epitopes present on specific cells shown in Table 2. The number of positive-stained cells in 3 consecutive areas of the valve grafts (leaflet, hinge area, and vascular wall) were scored by using an ocular grid allowing cell counts per 0.1 mm2. Analysis of nonspecific tissue reactions near the sutures was avoided.


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  		Table 2. Immunohistochemical Analysis of Aortic Valve Grafts

 
ConA stimulation and HTLf analysis
Peripheral blood mononuclear cells were isolated from the peripheral blood by Ficoll-Hypaque (Amersham Biotech AB, Uppsala, Sweden) density gradient and immediately used for in vitro tests. Recipient or donor spleen cells were obtained by mechanical dissociation of the spleen and Ficoll-Hypaque density gradient.

Peripheral blood mononuclear cells and spleen cells from all recipient animals were used for conA stimulation tests. Triplicate cultures of 105 PBMC or 5.104 spleen cells were incubated for 72 hours at 37° C and 5% CO2 in 96-well, round-bottomed microtiter plates (Nunclon, Roskilde, Denmark) containing culture medium (RPMI 1640 supplemented with 2 mmol/L L-glutamine, 10 mmol/L HEPES solution, 5.10-5 mol/L 2-mercaptoethanol, 100 U/mL penicillin, 100 mg/mL streptomycin and 10% heat inactivated fetal bovine serum) with or without 10 mg/mL of conA (Amersham Biotech AB). To each well, 0.5 µCi of 3H-thymidine (Amersham Biotech AB) was added 8 hours before harvesting. Incorporated 3H-Thymidine was measured in a scintillation spectrophotometer (Betaplate 1205; LKB-Wallac, Turku, Finland). The stimulation index (SI) was calculated by the formula: SI = mean counts per minute (cpm) with conA divided by mean cpm without conA.

Helper T lymphocyte frequencies were calculated by limiting dilution assay of PBMC and spleen cells from each experimental animal. Twelve replicate cultures were set up for six dilutions of responder cells (PBMC: 1.105 to 1563 per well; spleen cells: 5.104 to 781 per well) from recipients origin in 96-well U-bottomed microtiter plates containing 5.104 per well irradiated (30 Gy) splenic donor or autologous cells. After incubation in culture medium for 72 hours at 37° C and 5% CO2, 100 µl supernatant of each well was transferred to new U-bottomed plates. Functional interleukin-2 (IL-2) in the supernatant, produced by the responding HTLfs were measured and calculated using an IL-2 bioassay [6]. Frequency estimates are presented as the number of helper T-lymphocytes per million PBMC or spleen cells [6].

Statistical analysis
Differences between the experimental groups were tested with the Mann-Whitney U test using InStat software (GraphPad, San Diego, CA). The limit of significance was set on a two-tailed p value less than 0.05.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Functional and macroscopic evaluation
Three animals were excluded from the study because of death by hemorrhage or obliteration of the graft. Syngeneic grafts all remained competent after transplantation, whereas fresh allogeneic valve grafts progressively lost their competence. Early valve incompetence was observed in the cryopreservation groups. Thickening of valve leaflet, aortic wall, and sinus thrombosis was more often seen among allografts (Table 1).


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  		Table 1. Valve Competence and Histomorphology of Aortic Valve Grafts

 
Histology and immunohistochemistry
Syngeneic grafts demonstrated minor infiltration of mononuclear cells in the leaflet and adventitial layer at day 7 that was diminished by day 21. Progressive (moderate to severe) infiltration of (CD8+) T cells, macrophages, and dendritic cells was prominent in the adventitial layer spreading to the medial and intimal layer of allografts. Allogeneic valve leaflets were infiltrated by (CD8+) T cells and macrophages leading to complete loss of stromal cells and distorted leaflet structure at day 21 (Figs 1A, 1B). Cryopreserved syngeneic grafts showed prolonged influx of T-cells and macrophages, whereas cryopreserved allografts were infiltrated by mononuclear cells similar to their fresh counterparts. Progressive reduction of the numbers of cells stained positively for donor MHC class II antigens was observed in the valve leaflet and the hinge area of allografts (Figs 2A, 2B). Cryopreservation appeared to decreased the amount of donor derived cells (Fig 2C) (See Tables 1 and 2).



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  		Fig 1. Light-microscopic photographs of longitudinal sections of fresh aortic valve allograft (WAG to DA) at 7 days (A) and 21 days (B) after implantation (100x, hematoxylin-eosin stained). Valve leaflet (arrow) is heavily infiltrated by mononuclear cells at day 7, which resulted in dysmorph, acellular leaflets at day 21. Adventitial wall thickening, infiltration, and media cell loss were progressive in time.

 


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  		Fig 2. Light-microscopic photographs of immunohistochemical stained (anti-OX3, WAG MHC class II antigen) sections of fresh allogeneic valve grafts (WAG to DA) at 7 days (A) and 21 days (B) after implantation (100x). Reduction of areas consisting of cells positive for OX3 is seen in time (arrows), indicating loss of donor derived MHC class II positive cells. (C) OX3 expression (arrow) of cryopreserved valve allograft at day 7 after implantation illustrating early reduction of donor derived cells compared to fresh counterparts. (L = valve leaflet; MHC = major histocompatibility complex.)

 
Concanavalin A stimulation and HTLf
ConA stimulation (Fig 3) indices of all cell samples exceeded 25, which indicated the viability and immune reactivity of the cell samples. At day 21, spleen cells from allograft recipients had a higher SI compared to syngeneic graft recipients (p < 0.01), whereas cryopreservation of the graft reduced the reactivity of graft recipients (CA < FA). In allograft recipients there was progressive increased reactivity among PBMC (p < 0.01). By day 21, allograft recipients showed elevated SIs within PBMC again, whereas the SIs of isograft recipients returned to their preoperative values (preoperative median SI = 40.0, p < 0.002).



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  		Fig 3. Frequency of donor-reactive Helper T lymphocytes (HTL) (left scatters, left Y-axes) and concanavalin A stimulation indices (right bar, right Y-axes) among peripheral blood mononuclear cells (PBMC) (A, B) or spleen cells (C,D) of different aortic valve recipients at different time points. (CA = cryopreserved allograft; ConA SI = concanavalin A stimulation; CS = cryopreserved syngeneic graft; FA = fresh allograft; FS = fresh syngeneic graft; 7 = seventh post transplantation day; 21 = twenty first post transplantation day; Horizontal bars = resemble median value and range.)

 
Helper T lymphocyte frequencies (Fig 3) were higher among spleen cells of allograft recipients compared to isograft recipients at both 7 and 21 days (p < 0.05). In peripheral blood, increased HTLf was only seen at day 21 among allograft recipients. Recipients of fresh allografts gained higher frequencies compared to cryopreserved allografts (p < 0.03).


   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
The results presented in this study demonstrate that fresh aortic valve allografts implanted heterotopically in rats exhibit early histopathologic changes characteristic for rejection. Our findings are in agreement with other comparable studies suggesting that immune-mediated processes cause valve allograft deterioration [4, 7]. Interestingly, intense and progressive infiltration of T lymphocytes, macrophages and dendritic cells in the allogeneic valve leaflets (day 7) did not result in valve dysfunction. Valve incompetence was found later at day 21 and was accompanied by acellular and structural distorted leaflets, illustrating that early cellular infiltration can lead to structural valve deterioration and dysfunction. Phenotyping the infiltrates revealed early involvement of CD8+ T lymphocytes suggesting T cells to be the primarily effector cells initiating and regulating the donor-specific immune response. The influx of macrophages in the allograft is progressive and could be stimulated by local expression of cytokines and chemokines by activated T cells. It was found that the number of dendritic cells had increased in allografts, whereas only a few donor major histocompatibility complex class II positive cells were seen. We suggest that the dendritic cells involved in this allo-response are recruited from the recipient.

The histomorphologic changes of allografts are accompanied by an antidonor immune response detected in the spleen and peripheral blood. T-cell reactivity measured by conA stimulation revealed early increase of SI among PBMCs probably caused by a nonspecific activation after surgical intervention. At day 21 the SI of syngeneic graft recipients returned to a normal value (SI = 40), whereas the value remained high in allograft recipients because of persistent allostimulation. Among the splenocytes, a similar difference in the SI was seen, which suggests an increase of activated T cells in the secondary lymphoid organs. The analysis of Helper T lymphocyte frequencies in the spleen demonstrates increased allo-reactive HTLf among splenocytes already at day 7. These findings correspond with the study performed by Zhao and colleagues [7], who also identified an increase of donor specific cytotoxic T cells after valve allograft implantation in rats. However, their experiments did not include analysis of immune competent cells in peripheral circulation. In our study, the donor-reactive HTLfs increase among PBMCs were observed at a later time, indicating a sequence of primary activation of HTL in lymphoid organs followed by secondary release of activated HTL in the peripheral blood.

Histopathologic differences between cryopreserved syngeneic and allogeneic valve grafts were comparable to their fresh counterparts. However, sinus thrombosis, medial cell loss, and intima thickening were more prominent in the cryopreserved valves and these were caused by the cytotoxic effect and physical surface damage of cryopreservation [4, 8]. In addition, cryopreservation of the allografts appeared to reduce the antigenicity of valve grafts, shown by a significant lower conA reactivity and HTLfs in both spleen and PBMCs compared with fresh allograft recipients (p < 0.03). Nevertheless, cryopreserved allografts maintain their capacity to induce an immune response within the recipients indicated by higher antidonor T-cell reactivity compared with syngeneic graft recipients (p < 0.03).

In this study, we have demonstrated that valve allografts are able to provoke a donor-reactive immune response within the recipient resulting in graft destruction and dysfunction. Cryopreservation of valve allografts not only diminishes the antigenicity but they also alter their structural nature leading to additional nonimmunological degeneration. The increase of HTLf in both spleen and peripheral blood after allogeneic transplantation corresponds with histopathologic signs of rejection in the graft and therefore, confirms the important role of helper T cells in the immune response underlying allograft rejection [9]. Therefore, we suggest that clinical monitoring of helper T cell frequencies in peripheral blood of valve allograft recipients could serve as a noninvasive tool for identifying cellular rejection of the transplanted valve.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
The authors thank Frank Dor and Sandra van den Engel for in vitro testing and immunohistochemical staining. This study was financially supported in part by the Netherlands Heart Foundation (grant no.: 96.177).


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Hoekstra F., Knoop C., Jutte N., et al. Effect of cryopreservation and HLA-DR matching on the cellular immunogenicity of human cardiac valve allografts. J Heart Lung Transplant 1994;13:1095-1098.[Medline]
  2. Oei F.B., Welters M.J., Vaessen L.M., Stegmann A.P., Bogers A.J., Weimar W. Induction of cytotoxic T lymphocytes with destructive potential after cardiac valve homograft implantation. J Heart Valve Dis 2000;9:761-768.[Medline]
  3. O’Brien M.F., Stafford E.G., Gardner M.A., Pohlner P.G., McGiffin D.C. A comparison of aortic valve replacement with viable cryopreserved and fresh allograft valves, with a note on chromosomal studies. J Thorac Cardiovasc Surg 1987;94:812-823.[Abstract]
  4. Moustapha A., Ross D.B., Bittira B., et al. Aortic valve grafts in the rat: evidence for rejection. J Thorac Cardiovasc Surg 1997;114:891-902.[Abstract/Free Full Text]
  5. Oei F.B., Welters M.J., Vaessen L.M., Marquet R.L., Weimar W., Bogers A.J. Heart valve dysfunction due to cellular rejection in a novel heterotopic transplantation rat model. Transpl Int 2000;13:S528-S531.
  6. Vaessen L.M., Daane C.R., Maat A.P., Balk A.H., Claas F.H., Weimar W. T helper frequencies in peripheral blood reflect donor-directed reactivity in the graft after clinical heart transplantation. Clin Exp Immunol 1999;118:473-479.[Medline]
  7. Zhao X.M., Green M., Frazer I.H., Hogan P., O’Brien M.F. Donor-specific immune response after aortic valve allografting in the rat. Ann Thorac Surg 1994;57:1158-1163.[Abstract]
  8. Legare J.F., Lee T.D., Ross D.B. Cryopreservation of rat aortic valves results in increased structural failure. Circulation 2000;102:SIII75-SIII78.[Abstract/Free Full Text]
  9. Hall B.M. Cells mediating allograft rejection. Transplantation 1991;51:1141-1151.[Medline]



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