Alwayn I P, Buhler L, Appel J Z, Goepfert C, Csizmadia E, Correa L, Harper D, Kitamura H, Down J, Awwad M, Sackstein R, Cooper D K, Robson S C
The Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA.
Transplantation. 2001 Jun 15;71(11):1601-9. doi: 10.1097/00007890-200106150-00020.
Attempts to induce tolerance though mixed hematopoietic chimerism in the discordant pig-to-baboon xenotransplantation model are sometimes complicated by a potentially fatal thrombotic microangiopathy in the recipient baboons. This state develops immediately after the infusion of porcine mobilized peripheral blood leukocytes, containing progenitor cells (PBPC). In our study, we examined the interaction of infused porcine PBPC with recipient platelets in vivo in baboons and investigated the underlying mechanisms using an in vitro model.
Two naïve baboons and six baboons preconditioned with irradiation and immunosuppression that received porcine PBPC were evaluated in vivo. The interaction of porcine and baboon PBPC with baboon platelets was investigated by an in vitro platelet aggregation assay. Fresh and cryopreserved PBPC were evaluated as well as PBPC obtained from growth-factor mobilized and unmobilized pigs. Furthermore, cellular subsets of PBPC were assessed for potential to induce platelet aggregation. Immunohistochemical staining was performed on platelet-leukocyte aggregates and potential inhibition of aggregation with anti-P-selectin and anti-CD154 mAbs, or eptifibatide (a GPIIb/IIIa receptor antagonist), was tested.
All baboons that received porcine PBPC rapidly developed marked thrombocytopenia (<20,000/microl), elevated serum lactate dehydrogenase (>1,500U/liter), schistocytosis, and platelet aggregates on blood smear. Three baboons died (two untreated and one preconditioned), and substantive platelet aggregates containing porcine leukocytes were observed in the microvasculature of lungs and kidneys. In vitro, porcine, but not baboon, PBPC induced aggregation of baboon platelets in a dose-dependent manner. Immunohistological examination of these aggregates confirmed the incorporation of porcine leukocytes. Cryopreserved PBPC caused less aggregation than fresh PBPC, and growth-factor-mobilized PBPC induced less aggregation than unmobilized PBPC. Aggregation was fully abrogated by the addition of eptifibatide, and modulated by anti-P-selectin and anti-CD154 monoclonal antibodies that recognize adhesion receptors on activated platelets. Purified fractions (granulocytes, CD2+, and CD- cells) of porcine PBPC did not initiate aggregation, whereas addition of exogenous porcine PBPC membranes (erythrocytes, dead cells, and/or platelets) to the purified fractions exacerbated the aggregation response.
These data indicate that porcine PBPC mediate aggregation of baboon platelets. This process likely contributes to the thrombotic microangiopathy observed after PBPC transplantation in the pig-to-baboon model. Eptifibatide can fully abrogate platelet aggregation induced by porcine PBPC in vitro. Purification of the progenitor cells from porcine PBPC and/or treatment of baboons with eptifibatide may be beneficial.
在不匹配的猪-狒狒异种移植模型中，试图通过混合造血嵌合体诱导免疫耐受有时会因受体狒狒出现潜在致命的血栓性微血管病而变得复杂。这种状态在输注含有祖细胞（PBPC）的猪动员外周血白细胞后立即出现。在我们的研究中，我们在狒狒体内研究了输注的猪PBPC与受体血小板的相互作用，并使用体外模型研究了潜在机制。
对两只未经处理的狒狒和六只经照射和免疫抑制预处理并接受猪PBPC的狒狒进行体内评估。通过体外血小板聚集试验研究猪和狒狒PBPC与狒狒血小板的相互作用。对新鲜和冷冻保存的PBPC以及从生长因子动员和未动员的猪获得的PBPC进行了评估。此外，评估了PBPC的细胞亚群诱导血小板聚集的潜力。对血小板-白细胞聚集体进行免疫组织化学染色，并测试抗P-选择素和抗CD154单克隆抗体或依替巴肽（一种GPIIb/IIIa受体拮抗剂）对聚集的潜在抑制作用。
所有接受猪PBPC的狒狒迅速出现明显的血小板减少（<20,000/微升）、血清乳酸脱氢酶升高（>1,500U/升）、裂体细胞增多以及血涂片上出现血小板聚集体。三只狒狒死亡（两只未治疗，一只预处理），在肺和肾的微血管中观察到含有猪白细胞的大量血小板聚集体。在体外，猪PBPC而非狒狒PBPC以剂量依赖方式诱导狒狒血小板聚集。对这些聚集体的免疫组织学检查证实了猪白细胞的掺入。冷冻保存的PBPC引起的聚集比新鲜PBPC少，生长因子动员的PBPC诱导的聚集比未动员的PBPC少。加入依替巴肽可完全消除聚集，而抗P-选择素和抗CD154单克隆抗体可调节聚集，这些抗体可识别活化血小板上的粘附受体。猪PBPC的纯化组分（粒细胞、CD2+和CD-细胞）不会引发聚集，而向纯化组分中加入外源性猪PBPC膜（红细胞、死细胞和/或血小板）会加剧聚集反应。
这些数据表明猪PBPC介导狒狒血小板聚集。这一过程可能导致猪-狒狒模型中PBPC移植后观察到的血栓性微血管病。依替巴肽可在体外完全消除猪PBPC诱导的血小板聚集。从猪PBPC中纯化祖细胞和/或用依替巴肽治疗狒狒可能有益。
