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Cellular immunity overrules the protective effect of human DAF as demonstrated in an ex vivo heart perfusion model
Cellular Immunity Overrules the Protective Effect of Human DAF as Demonstrated in an Ex Vivo Heart Perfusion Model C.A.E. Verbakel, I. Anegon, S. Me´noret, R.L. Marquet, and J.N.M. Ijzermans
T
HE USE OF donors transgenic for recipient-type complement regulatory proteins, such as Decay Accelerating Factor (DAF), protect grafts against hyperacute rejection (HAR). HAR is primary mediated by xenoreactive natural antibodies and complement. We demonstrated earlier that rat hearts transgenic for human DAF (hDAF), perfused with human serum, were protected against HAR for more than 60 minutes.1 Although the hearts were not rejected by HAR, they were finally rejected by an acute vascular rejection (AVR) process. The remaining question is whether hDAF transgenic hearts are protected against cell-mediated immunity as well. Therefore, the aim of our study was to analyze this process in an ex vivo rat perfusion model, the Langendorff device. MATERIALS AND METHODS Animals hDAF transgenic rats were bred by using a construct containing the hDAF cDNA under the transcriptional control of the endothelial cell-specific human ICAM-2 promoter, as described by Charreau et al.1 The animals were housed in a certified animal breeding facility under standard conditions and had free access to standard rat chow and water acidified to pH 2.2 ad libitum. The experimental protocol was approved by the committee on Animal Research of the Erasmus University. Wistar rat hearts were used as controls. The experimental groups consisted of six controls and six hDAF transgenes, perfused with 12% human blood, and eight controls and eight hDAF transgenes, perfused with 12% heat-inactivated human blood.
Surgical Procedure and ex Vivo Perfusion Rats were anesthetized with isoflurane inhalation and 1.0 mL Heparin (50 IU/mL) was injected intravenously. Via two lateral incisions the thorax was opened and the heart was removed. A 14-gauge in-dwelling cannula was inserted into the aorta and secured with a 4.0 ligature. The heart was contiguously perfused with oxygenated Krebs-Henseleit solution using a syringe and directly linked to the Langendorff circuit. All hearts were perfused with Krebs-Henseleit solution for 10 minutes and were then switched to perfusion with 12% (heat-inactivated) human blood for 60 minutes.
Human Serum/Blood Frozen, pooled, human O-type plasma was obtained from the blood bank of the University Hospital Rotterdam. For each © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
experiment fresh human serum was prepared from thawed human plasma after the induction of clotting with 0.5 mL thrombin/200 mL human plasma. Heat inactivation, ie, complement inactivation, was realized by heating of the serum at 56° for 30 minutes. O-type blood was collected in 9-mL EDTA test tubes and immediately processed as follows. After centrifugation, the plasma was removed and replaced by a similar volume of serum, which was prepared as described above. To prepare the 12% solution for perfusion, the blood was diluted with Krebs-Henseleit stock solution.
Immunohistochemistry By means of standard histology (H&E) and immunohistochemistry (anti-human CD45, P-selectin, and ICAM), hearts were analyzed for differences in amounts of leukocytes adhering to the endothelium. Twenty vessels were examined to evaluate the presence of leukocytes. In each vessel the amount of leukocytes adhering to the endothelium and the amount of leukocytes present in the lumen were counted. Finally, the percentage adherence was calculated. Because adhesion of leukocytes is mediated by the early adhesion molecules P-selectin and ICAM, we looked at the expression of these adhesion molecules on the endothelium and analyzed them semiquantitatively.
Statistical Analysis Student’s t test was used to evaluate the difference in cell adhesion between groups and was considered significant when P ⬍ .05.
RESULTS AND DISCUSSION
Mean survival times of the hearts are summarized in Table 1. H&E-stained transgenic hearts perfused with heat-inactivated human blood showed significantly (P ⫽ .001) fewer adhering leukocytes to the endothelium, and immunohistochemistry, using antihuman CD45, showed significantly (P ⫽ .000) fewer leukocyte-endothelium interactions as compared to controls. Although H&E-stained transgenic hearts, perfused with human blood, showed significantly From the Laboratory for Experimental Surgery, Erasmus University (C.A.E.V., R.L.M., J.N.M.I.), Rotterdam, The Netherlands, and INSERM U437 “Immunointervention en Allo et Xe´notransplantation” and Institut de Transplantation et de Recherche en Transplantation (I.A., S.M.), Nantes, France. Supported by a grant of the Netherlands Heart Foundation. Address reprint requests to C.A.E. Verbakel, Laboratory for Experimental Surgery, Erasmus University Rotterdam, Room Ee102c, PO Box 1738, 3000 DR Rotterdam, The Netherlands. 0041-1345/01/$–see front matter PII S0041-1345(00)02252-1 781
Transplantation Proceedings, 33, 781–782 (2001)
VERBAKEL, ANEGON, ME´NORET ET AL
782 Table 1. Mean Survival Times (MSTs) and Percentage of Leukocytes Adhering to Vessel Wall in hDAF Transgenic Rat Hearts and Wistar (W) Controls Perfused with Heat-Inactivated (HI) Human Blood (HB) in the Langendorff System Group
6 6 8 8
W ⫹ 12% HB hDAF ⫹ 12% HB W ⫹ 12% HI-HB hDAF ⫹ 12% HI-HB
MSTs (minutes)
Percentage Adhering Cells (H&E/CD45)
19 51* 34 38
66.1 ⫾ 14*/65.7 ⫾ 18 39.4 ⫾ 11*/58 ⫾ 11 53.3 ⫾ 14*/65.6 ⫾ 6* 28.5 ⫾ 9*/32 ⫾ 15*
*P ⬍ .05.
(P ⫽ .004) fewer adhering leukocytes to the endothelium as compared to controls, antihuman CD45-stained hDAF hearts showed no difference (P ⫽ .392) in leukocyte-
endothelium interactions. The intensity of ICAM expression was the same in all groups, whereas the intensity of the expression of P-selectin was lower in the hDAF hearts perfused with human blood. Apparently a delay in the activation of endothelium occurred in this group, leading to a lower expression of P-selectin and a decline in the number of leukocyte-endothelium interactions. In conclusion, blood cells may contribute to rejection of nontransgenic as well as transgenic hearts. Thus, hDAF may not be sufficient to prevent AVR, despite its inhibitory effect on leukocyte adhesion.
REFERENCE 1. Charreau B, Me´noret S, Tesson L, et al: Mol Med 5:617, 1999
T
HE USE OF donors transgenic for recipient-type complement regulatory proteins, such as Decay Accelerating Factor (DAF), protect grafts against hyperacute rejection (HAR). HAR is primary mediated by xenoreactive natural antibodies and complement. We demonstrated earlier that rat hearts transgenic for human DAF (hDAF), perfused with human serum, were protected against HAR for more than 60 minutes.1 Although the hearts were not rejected by HAR, they were finally rejected by an acute vascular rejection (AVR) process. The remaining question is whether hDAF transgenic hearts are protected against cell-mediated immunity as well. Therefore, the aim of our study was to analyze this process in an ex vivo rat perfusion model, the Langendorff device. MATERIALS AND METHODS Animals hDAF transgenic rats were bred by using a construct containing the hDAF cDNA under the transcriptional control of the endothelial cell-specific human ICAM-2 promoter, as described by Charreau et al.1 The animals were housed in a certified animal breeding facility under standard conditions and had free access to standard rat chow and water acidified to pH 2.2 ad libitum. The experimental protocol was approved by the committee on Animal Research of the Erasmus University. Wistar rat hearts were used as controls. The experimental groups consisted of six controls and six hDAF transgenes, perfused with 12% human blood, and eight controls and eight hDAF transgenes, perfused with 12% heat-inactivated human blood.
Surgical Procedure and ex Vivo Perfusion Rats were anesthetized with isoflurane inhalation and 1.0 mL Heparin (50 IU/mL) was injected intravenously. Via two lateral incisions the thorax was opened and the heart was removed. A 14-gauge in-dwelling cannula was inserted into the aorta and secured with a 4.0 ligature. The heart was contiguously perfused with oxygenated Krebs-Henseleit solution using a syringe and directly linked to the Langendorff circuit. All hearts were perfused with Krebs-Henseleit solution for 10 minutes and were then switched to perfusion with 12% (heat-inactivated) human blood for 60 minutes.
Human Serum/Blood Frozen, pooled, human O-type plasma was obtained from the blood bank of the University Hospital Rotterdam. For each © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
experiment fresh human serum was prepared from thawed human plasma after the induction of clotting with 0.5 mL thrombin/200 mL human plasma. Heat inactivation, ie, complement inactivation, was realized by heating of the serum at 56° for 30 minutes. O-type blood was collected in 9-mL EDTA test tubes and immediately processed as follows. After centrifugation, the plasma was removed and replaced by a similar volume of serum, which was prepared as described above. To prepare the 12% solution for perfusion, the blood was diluted with Krebs-Henseleit stock solution.
Immunohistochemistry By means of standard histology (H&E) and immunohistochemistry (anti-human CD45, P-selectin, and ICAM), hearts were analyzed for differences in amounts of leukocytes adhering to the endothelium. Twenty vessels were examined to evaluate the presence of leukocytes. In each vessel the amount of leukocytes adhering to the endothelium and the amount of leukocytes present in the lumen were counted. Finally, the percentage adherence was calculated. Because adhesion of leukocytes is mediated by the early adhesion molecules P-selectin and ICAM, we looked at the expression of these adhesion molecules on the endothelium and analyzed them semiquantitatively.
Statistical Analysis Student’s t test was used to evaluate the difference in cell adhesion between groups and was considered significant when P ⬍ .05.
RESULTS AND DISCUSSION
Mean survival times of the hearts are summarized in Table 1. H&E-stained transgenic hearts perfused with heat-inactivated human blood showed significantly (P ⫽ .001) fewer adhering leukocytes to the endothelium, and immunohistochemistry, using antihuman CD45, showed significantly (P ⫽ .000) fewer leukocyte-endothelium interactions as compared to controls. Although H&E-stained transgenic hearts, perfused with human blood, showed significantly From the Laboratory for Experimental Surgery, Erasmus University (C.A.E.V., R.L.M., J.N.M.I.), Rotterdam, The Netherlands, and INSERM U437 “Immunointervention en Allo et Xe´notransplantation” and Institut de Transplantation et de Recherche en Transplantation (I.A., S.M.), Nantes, France. Supported by a grant of the Netherlands Heart Foundation. Address reprint requests to C.A.E. Verbakel, Laboratory for Experimental Surgery, Erasmus University Rotterdam, Room Ee102c, PO Box 1738, 3000 DR Rotterdam, The Netherlands. 0041-1345/01/$–see front matter PII S0041-1345(00)02252-1 781
Transplantation Proceedings, 33, 781–782 (2001)
VERBAKEL, ANEGON, ME´NORET ET AL
782 Table 1. Mean Survival Times (MSTs) and Percentage of Leukocytes Adhering to Vessel Wall in hDAF Transgenic Rat Hearts and Wistar (W) Controls Perfused with Heat-Inactivated (HI) Human Blood (HB) in the Langendorff System Group
6 6 8 8
W ⫹ 12% HB hDAF ⫹ 12% HB W ⫹ 12% HI-HB hDAF ⫹ 12% HI-HB
MSTs (minutes)
Percentage Adhering Cells (H&E/CD45)
19 51* 34 38
66.1 ⫾ 14*/65.7 ⫾ 18 39.4 ⫾ 11*/58 ⫾ 11 53.3 ⫾ 14*/65.6 ⫾ 6* 28.5 ⫾ 9*/32 ⫾ 15*
*P ⬍ .05.
(P ⫽ .004) fewer adhering leukocytes to the endothelium as compared to controls, antihuman CD45-stained hDAF hearts showed no difference (P ⫽ .392) in leukocyte-
endothelium interactions. The intensity of ICAM expression was the same in all groups, whereas the intensity of the expression of P-selectin was lower in the hDAF hearts perfused with human blood. Apparently a delay in the activation of endothelium occurred in this group, leading to a lower expression of P-selectin and a decline in the number of leukocyte-endothelium interactions. In conclusion, blood cells may contribute to rejection of nontransgenic as well as transgenic hearts. Thus, hDAF may not be sufficient to prevent AVR, despite its inhibitory effect on leukocyte adhesion.
REFERENCE 1. Charreau B, Me´noret S, Tesson L, et al: Mol Med 5:617, 1999