MacDonald Shirley L, Downing Ian, Turner Marc, Kilpatrick David C
Scottish National Blood Transfusion Service, National Science Laboratory, Edinburgh, UK.
Biochem Soc Trans. 2008 Dec;36(Pt 6):1497-500. doi: 10.1042/BST0361497.
MBL (mannan-binding lectin; also called mannose-binding lectin) is a circulating C-type lectin with a collagen-like region synthesized mainly by the liver. MBL may influence susceptibility to infection in recipients of stem cell transplants, and it has even been suggested that the MBL status of a donor can influence the recipient's susceptibility to post-transplant infections. We have previously reported that MBL can be detected on human monocytes and monocyte-derived dendritic cells, based on detection using biotinylated anti-MBL, suggesting that those cells could synthesize MBL. If true, permanent MBL replacement therapy could be achieved by stem cell infusions. However, two other groups independently failed to find mbl-2-derived mRNA in monocytes. Therefore, to confirm or refute our previous observations, we used an alternative experimental strategy. Instead of using biotinylated antibody and labelled streptavidin, detection of surface MBL was attempted using MBL-specific primary antibodies (131-1, 131-10 and 131-11) followed by fluorescein-labelled anti-IgG, and controlled by the use of non-specific IgG as primary antibody. Monocytes were counterstained with anti-CD14-PE before FACS analysis. Adherent monocytes were also cultured for 48 h in serum-free medium or converted into immature dendritic cells by culture with IL-4 (interleukin-4) and GM-CSF (granulocyte/monocyte colony-stimulating factor). During FACS analysis, the dendritic cells were gated after counter-staining with anti-CD1a-PE. MBL was readily detected on the surface of fresh monocytes using all three specific anti-MBL monoclonal antibodies, but specific anti-MBL binding was greatly diminished after monocytes had been cultured for 2 days in serum-free medium. Moreover, we could not detect any MBL present on the surface of monocyte-derived dendritic cells. We therefore conclude that MBL is indeed present on the surface of fresh human monocytes. However, in view of the mRNA findings of others and our own previous observation that no secretion of MBL took place in culture, we presume that the surface-bound MBL is derived from autologous plasma and not synthesized by the cells. This conclusion is consistent with our in vivo findings in stem cell transplant patients which provided evidence against significant extra-hepatic production of serum MBL. It provides no ready explanation for the remarkable observation of Mullighan, Heatley, Doherty, Szabo, Grigg, Hughes, Schwarer, Szer, Tait, Bik To and Bardy [(2002) Blood 99, 3524-3529] that the presence of variant alleles of mbl-2 in stem cell donors can influence susceptibility to serious infections in their recipients.
甘露聚糖结合凝集素（MBL；也称为甘露糖结合凝集素）是一种循环的C型凝集素，具有主要由肝脏合成的胶原样区域。MBL可能会影响干细胞移植受者对感染的易感性，甚至有人提出供体的MBL状态会影响受者移植后感染的易感性。我们之前曾报道，基于使用生物素化抗MBL进行检测，在人单核细胞和单核细胞衍生的树突状细胞上可检测到MBL，这表明这些细胞可能合成MBL。如果属实，通过干细胞输注可实现永久性MBL替代疗法。然而，另外两个研究小组独立地未能在单核细胞中发现mbl - 2衍生的mRNA。因此，为了证实或反驳我们之前的观察结果，我们采用了另一种实验策略。不再使用生物素化抗体和标记的链霉亲和素，而是尝试使用MBL特异性一抗（131 - 1、131 - 10和131 - 11）检测表面MBL，随后使用荧光素标记的抗IgG，并以非特异性IgG作为一抗进行对照。在进行荧光激活细胞分选（FACS）分析之前，用抗CD14 - 藻红蛋白（PE）对单核细胞进行复染。贴壁单核细胞也在无血清培养基中培养48小时，或通过与白细胞介素 - 4（IL - 4）和粒细胞/单核细胞集落刺激因子（GM - CSF）培养转化为未成熟树突状细胞。在FACS分析过程中，用抗CD1a - PE复染后对树突状细胞进行门控。使用所有三种特异性抗MBL单克隆抗体均能在新鲜单核细胞表面轻松检测到MBL，但单核细胞在无血清培养基中培养2天后，特异性抗MBL结合显著减少。此外，我们在单核细胞衍生的树突状细胞表面未检测到任何MBL。因此，我们得出结论，MBL确实存在于新鲜人单核细胞表面。然而，鉴于其他人的mRNA研究结果以及我们自己之前观察到培养中未发生MBL分泌，我们推测表面结合的MBL源自自体血浆而非细胞合成。这一结论与我们在干细胞移植患者中的体内研究结果一致，该研究结果提供了反对血清MBL显著肝外产生的证据。对于Mullighan、Heatley、Doherty、Szabo、Grigg、Hughes、Schwarer、Szer、Tait、Bik To和Bardy [(2002) Blood 99, 3524 - 3529]的显著观察结果，即干细胞供体中mbl - 2变异等位基因的存在会影响其受者对严重感染的易感性，这一结论并未提供现成的解释。
