Gewirtz A M, Zhang J, Ratajczak J, Ratajczak M, Park K S, Li C, Yan Z, Poncz M
Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, USA.
Blood. 1995 Oct 1;86(7):2559-67.
We have previously shown that platelet factor 4 (PF4), a platelet-specific CXC chemokine, can directly and specifically inhibit human megakaryocyte colony formation. We therefore hypothesized that PF4 might function as a negative autocrine regulator of megakaryocytopoiesis. Herein we present additional studies characterizing the inhibitory effect of CXC chemokines on human megakaryocyte development. We first corroborated our initial studies by showing that recombinant human (rH) PF4, like the native protein, inhibited megakaryocytopoiesis. We then examined the inhibitory properties of other CXC family members. Neutrophil activating peptide-2 (NAP-2), a naturally occurring N-terminally cleaved beta TG peptide, was found to inhibit megakaryocytopoiesis with two to three orders of magnitude greater potency than PF4. Structure function studies showed that an N-terminal mutation, which eliminated NAP-2's neutrophil activating properties (NAP-2E2-->A), also abrogated its ability to inhibit megakaryocyte development. Further investigations of this type demonstrated that a chimeric PF4 protein (AELR/PF4) in which PF4's N-terminus was replaced with the first four amino acids of NAP-2 was also a potent inhibitor of megakaryocytopoiesis. Interleukin (IL)-8, another CXC chemokine, and three CC chemokines (macrophage inhibitory protein-1 alpha [MIP-1 alpha], MIP-1 beta, and C10) also specifically inhibited megakaryocyte colony formation at NAP-2 equivalent doses. CXC and CC chemokine inhibition was additive suggesting that the effects might be mediated through a common pathway. The inhibitory effects of NAP-2 and MIP-1 alpha could not be overcome by adding physiologically relevant amounts of recombinant human megakaryocyte growth and development factor (MGDR) (50 ng/mL) to the cultures. Using Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) based analyses, we documented mRNA expression of IL-8 receptor isoforms alpha and beta in total platelet RNA and in normal human megakaryocytes, respectively. Based on these results, we hypothesize that chemokines play a physiologic role in regulating megakaryocytopoiesis. Because chemokines are elaborated by ancillary marrow cells, both autocrine and paracrine growth control is suggested, the effects of which might be exerted, in part, through alpha and beta IL-8 receptors.
我们之前已经表明，血小板因子4（PF4），一种血小板特异性的CXC趋化因子，能够直接且特异性地抑制人巨核细胞集落形成。因此我们推测PF4可能作为巨核细胞生成的一种负性自分泌调节因子发挥作用。在此我们展示了另外一些研究，这些研究描述了CXC趋化因子对人巨核细胞发育的抑制作用。我们首先通过证明重组人（rH）PF4与天然蛋白一样能抑制巨核细胞生成，证实了我们最初的研究。然后我们检测了其他CXC家族成员的抑制特性。发现中性粒细胞激活肽-2（NAP-2），一种天然存在的N端裂解的βTG肽，抑制巨核细胞生成的效力比PF4高两到三个数量级。结构功能研究表明，一个消除了NAP-2中性粒细胞激活特性的N端突变（NAP-2E2→A），也消除了其抑制巨核细胞发育的能力。这类进一步的研究表明，一种嵌合PF4蛋白（AELR/PF4），其中PF4的N端被NAP-2的前四个氨基酸取代，也是巨核细胞生成的一种有效抑制剂。白细胞介素（IL）-8，另一种CXC趋化因子，以及三种CC趋化因子（巨噬细胞抑制蛋白-1α [MIP-1α]、MIP-1β和C10）在与NAP-2等效剂量时也能特异性抑制巨核细胞集落形成。CXC和CC趋化因子的抑制作用是相加的，这表明这些作用可能是通过一条共同途径介导的。向培养物中添加生理相关量的重组人巨核细胞生长和发育因子（MGDR）（50 ng/mL）并不能克服NAP-2和MIP-1α的抑制作用。使用基于Northern印迹和逆转录聚合酶链反应（RT-PCR）的分析，我们分别在总血小板RNA和正常人巨核细胞中记录了IL-8受体亚型α和β的mRNA表达。基于这些结果，我们推测趋化因子在调节巨核细胞生成中发挥生理作用。因为趋化因子是由辅助性骨髓细胞产生的，提示存在自分泌和旁分泌生长控制，其作用可能部分通过IL-8α和β受体来发挥。
