Frey B M, Rafii S, Crystal R G, Moore M A
Memorial Sloan-Kettering Cancer Center, New York, USA.
Schweiz Med Wochenschr. 1998 Oct 17;128(42):1587-92.
Using a new adenoviral vector (Ad) construct, we expressed human thrombopoietin (TPO) cDNA (AdTPO) in mice with various inherited immune deficiency syndromes such as nude, SCID and NOD-SCID mice. Immune normal Balb/c mice and a vector construct without TPOcDNA (AdNull), respectively, were used for controls. All animals (3 per group) were treated with a single application of 10(9) PFU (plaque forming unit) of Ad (AdTPO or AdNull) intraperitoneally on day 0. Four to 5 weeks following AdTPO administration, SCID and NOD-SCID mice demonstrated peak concentration of PLT of 12- to 14-fold normal value simultaneously with maximum concentration of PMNs (10- to 12-fold normal value). Later on these animals had a chronic thrombocytosis. In contrast, Balb/c mice and nude mice experienced PLT peak concentration of 4- to 6-fold normal value without granulocytosis 1 to 2 weeks following AdTPO treatment. Only nude mice had chronically elevated PLTs. In contrast, Balb/c mice developed thrombocytopenia due to cross-reacting anti-TPO antibodies. Animals with chronic thrombocytosis revealed increased content of CFU-G/GM, CFU-GEMM and CFU-Meg in bone marrow compared with controls. In contrast, Balb/c mice showed decreased content of CFUs if anti-TPO-antibodies were present. Histologically, only SCID mice developed severe osteomyelofibrosis and osteomyelosclerosis, hepato-splenomegaly, extramedullary hematopoiesis in liver and lung and ultimately suffered of progressive pancytopenia, anisocytosis, fragmentocytosis and a lethal wasting syndrome. In contrast, NOD-SCID mice which demonstrated similar extent of TPO overexpression and in addition to the B- and T-cellular immune deficiency harbour defective monocytes and macrophages, did not develop fibrotic changes of the bone marrow. From these results, we conclude (1) chronic TPO overexpression in vivo may lead to thrombocytosis and granulocytosis with expansion of CFU-GM, -GEMM and -Meg; (2) in vivo expression of adenovirally mediated TPOcDNA depends on immune competency of the host; (3) functionally normal monocytes and macrophages are indispensable for development of secondary osteomyelofibrosis and (4) adenovirally mediated expression of xenogeneic transgenes may brake immune tolerance for the respective self protein leading to autoimmune phenomena. Our in vivo model might provide further insights into the pathophysiology of secondary osteomyelofibrosis and may prove useful in designing new strategies for immune therapies of cancer.
我们使用一种新的腺病毒载体（Ad）构建体，在患有各种遗传性免疫缺陷综合征的小鼠（如裸鼠、严重联合免疫缺陷小鼠和非肥胖型糖尿病严重联合免疫缺陷小鼠）中表达人血小板生成素（TPO）cDNA（AdTPO）。分别使用免疫正常的Balb/c小鼠和不含TPO cDNA的载体构建体（AdNull）作为对照。所有动物（每组3只）在第0天腹腔内单次注射10⁹ PFU（噬斑形成单位）的Ad（AdTPO或AdNull）。在给予AdTPO后4至5周，严重联合免疫缺陷小鼠和非肥胖型糖尿病严重联合免疫缺陷小鼠的血小板浓度峰值达到正常值的12至14倍，同时中性粒细胞浓度达到最大值（正常值的10至12倍）。此后，这些动物出现慢性血小板增多症。相比之下，Balb/c小鼠和裸鼠在接受AdTPO治疗后1至2周，血小板浓度峰值达到正常值的4至6倍，且无粒细胞增多。只有裸鼠的血小板持续升高。相比之下，Balb/c小鼠因交叉反应性抗TPO抗体而出现血小板减少。与对照组相比，患有慢性血小板增多症的动物骨髓中CFU - G/GM、CFU - GEMM和CFU - Meg的含量增加。相反，如果存在抗TPO抗体，Balb/c小鼠的CFU含量会降低。组织学上，只有严重联合免疫缺陷小鼠出现严重的骨髓纤维化和骨髓硬化、肝脾肿大、肝脏和肺脏的髓外造血，最终发展为进行性全血细胞减少、红细胞大小不均、破碎红细胞增多和致命的消耗综合征。相比之下，非肥胖型糖尿病严重联合免疫缺陷小鼠虽然TPO过表达程度相似，且除了B细胞和T细胞免疫缺陷外还存在单核细胞和巨噬细胞缺陷，但并未出现骨髓纤维化改变。从这些结果中，我们得出以下结论：（1）体内慢性TPO过表达可能导致血小板增多症和粒细胞增多症，并伴有CFU - GM、-GEMM和-Meg的扩增；（2）腺病毒介导的TPO cDNA在体内的表达取决于宿主的免疫能力；（3）功能正常的单核细胞和巨噬细胞对于继发性骨髓纤维化的发展是不可或缺的；（4）腺病毒介导的异种转基因表达可能会破坏对相应自身蛋白的免疫耐受，导致自身免疫现象。我们的体内模型可能会为继发性骨髓纤维化的病理生理学提供进一步的见解，并可能在设计癌症免疫治疗的新策略方面证明是有用的。
