Activation of human endothelial cells by mobilized porcine leukocytes in vitro: Implications for mixed chimerism in xenotransplantation

Academic Article

Abstract

  • Background. The induction of immunologic tolerance to pig antigens in primates may facilitate the development of successful clinical xenotransplantation protocols. The infusion of mobilized porcine peripheral blood leukocytes (PBPC, consisting of approximately 2% peripheral blood progenitor cells) into preconditioned baboons, intended to induce mixed hematopoietic cell chimerism, however, results in a severe thrombotic microangiopathy (TM) that includes vascular injury, microvascular thrombosis, and pronounced thrombocytopenia. Because the mechanisms responsible for TM are unclear, we have explored the effects of PBPC on human umbilical vein endothelial cell (HUVEC) activation. Methods. Confluent HUVEC monolayers were established in 96-well cell culture clusters. PBPC were mobilized from miniature swine with porcine interleukin 3 (pIL-3), porcine stem cell factor (pSCF), and human granulocyte-colony stimulating factor (hG-CSF) and were collected by leukapheresis. PBPC were added to HUVEC (0-1×107 PBPC/well) for 3- to 24-hr periods and, with cell-based ELISA techniques, surface levels of E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) were measured. In some cases, peripheral blood leukocytes (PBL) were collected from pigs that did not receive pIL-3, pSCF, or hG-CSF and were added to HUVEC. PBPC were also sorted into subsets of CD2- cells, CD2+ cells, and cellular debris, each of which were added separately to HUVEC. Transwell permeable membrane inserts were placed over HUVEC to prevent direct cell-cell contact with PBPC in some instances Results. PBPC from different pigs (n=6) induced an increase in the expression of E-selectin, VCAM-1, and ICAM-1 to levels 5, 4, and 2 times greater than baseline, respectively. ICAM-1 expression reached maximum levels after the addition of 6×105 PBPC/well. Expression of E-selectin and VCAM-1 increased further with the addition of greater numbers of PBPC, reaching maximum levels after the addition of 1×107 PBPC/ well. PBPC-induced up-regulation of E-selectin, VCAM-1, and ICAM-1 had a maximum effect after approximately 6 hr, 12 hr, and 6 to 9 hr, respectively (n=3). The effects of fresh and frozen PBPC on HUVEC were similar (n=2). Compared to PBPC, PBL induced higher levels of E-selectin, VCAM-1, and ICAM-1 on HUVEC (n=2). The addition of CD2- cells to HUVEC induced an increase in E-selectin and VCAM-1 to levels 4 times greater than baseline, whereas the addition of CD2+ cells or debris did not elicit a substantial effect (n=2). Transwell permeable membranes prevented PBPC-induced up-regulation of E-selectin, VCAM-1, and ICAM-1 on HUVEC (n=2), suggesting that the mechanism of activation requires direct cell-cell contact. Conclusions. Porcine PBPC activate HUVEC, as suggested by an increase in surface E-selectin, VCAM-1, and ICAM-1 levels, and have a maximum effect after 9 hr. Freezing of PBPC does not affect PBPC-induced activation of HUVEC. PBL induce greater activation of HUVEC than do PBPC. CD2- cells are primarily responsible for PBPC-induced activation of HUVEC and direct cell-cell contact is required. Removal of CD2-cells before the administration of PBPC or the use of agents that interrupt PBPC-endothelial cell interactions may prevent or treat TM in baboons.
  • Authors

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    Author List

  • Appel JZ; Newman D; Awwad M; Kruger Gray HS; Down J; Cooper DKC; Robson SC
  • Start Page

  • 1302
  • End Page

  • 1309
  • Volume

  • 73
  • Issue

  • 8