Zhang Alexandra Y, Wu Caiyun, Zhou Lixin, Ismail Sahar A, Tao Jianming, McCormick Laura L, Cooper Kevin D, Gilliam Anita C
Department of Dermatology, Case/University Hospitals of Cleveland, OH 44106, USA.
Exp Dermatol. 2006 Jan;15(1):51-7. doi: 10.1111/j.0906-6705.2005.00394.x.
We have selectively targeted monocyte/macrophages overexpressing an immunomodulatory molecule, latency-associated peptide (LAP), a naturally occurring antagonist for transforming growth factor-beta1, to murine skin utilizing UV light to produce a cutaneous influx of transduced monocyte/macrophages. Bone marrow (BM) cells from BALB/c mice were transduced in vitro with a retroviral construct containing green fluorescent protein (GFP) for detection and human LAP (hLAP) as a test molecule. The transduced BM cells were then cultured in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) to produce differentiation to monocyte/macrophages. More than 80% of transduced BM cells were CD11b-positive and MOMA-positive by fluorescence-activated cell-sorter analysis and secreted LAP by ELISA after 10 days of culture in granulocyte-macrophage colony-stimulating factor (GM-CSF). Transduced monocyte/macrophages containing either GFP or hLAP-GFP were then injected intravenously into BALB/c mice. One-half of recipients in each group were exposed to UVB (72 mJ) to induce monocyte/macrophage infiltration into skin. Recipients were sacrificed 60 h after UV irradiation. We found transduced cutaneous macrophages expressing GFP by examining with fluorescence microscopy frozen skin sections of recipient mice immunostained with antibodies to GFP and to macrophage marker F4/80. We identified hLAP sequences by polymerase chain reaction (PCR) of total DNA in recipient blood and UV-irradiated skin but not in unirradiated skin. LAP sequences were also detected at much lower levels in other organs (lung, spleen, and liver) by PCR. Therefore, we have shown that genetically altered monocytic cells can be injected intravenously and targeted to mouse skin by UV exposure. This macrophage-based gene-transfer method may be a potentially useful immunotherapeutic approach for delivering monocyte/macrophage-derived products to skin.
我们利用紫外线将过表达免疫调节分子——潜伏相关肽(LAP,一种天然存在的转化生长因子-β1拮抗剂)的单核细胞/巨噬细胞选择性地靶向至小鼠皮肤,以促使转导的单核细胞/巨噬细胞向皮肤内流入。用含有绿色荧光蛋白(GFP)用于检测以及人LAP(hLAP)作为测试分子的逆转录病毒构建体在体外转导来自BALB/c小鼠的骨髓(BM)细胞。然后将转导的BM细胞与粒细胞-巨噬细胞集落刺激因子(GM-CSF)在体外共同培养,使其分化为单核细胞/巨噬细胞。通过荧光激活细胞分选分析,超过80%的转导BM细胞呈CD11b阳性和MOMA阳性,并且在粒细胞-巨噬细胞集落刺激因子(GM-CSF)中培养10天后通过酶联免疫吸附测定法分泌LAP。然后将含有GFP或hLAP-GFP的转导单核细胞/巨噬细胞静脉注射到BALB/c小鼠体内。每组中一半的受体接受紫外线B(72 mJ)照射,以诱导单核细胞/巨噬细胞浸润到皮肤中。紫外线照射60小时后处死受体小鼠。通过荧光显微镜检查受体小鼠的冷冻皮肤切片,该切片用抗GFP抗体和巨噬细胞标志物F4/80进行免疫染色,我们发现转导的皮肤巨噬细胞表达GFP。我们通过对受体血液和紫外线照射皮肤中的总DNA进行聚合酶链反应(PCR)鉴定出hLAP序列,但未在未照射的皮肤中鉴定出。通过PCR在其他器官(肺、脾和肝)中也检测到低得多水平的LAP序列。因此,我们已经表明,基因改造的单核细胞可以静脉注射,并通过紫外线照射靶向至小鼠皮肤。这种基于巨噬细胞的基因转移方法可能是一种潜在有用的免疫治疗方法,用于将单核细胞/巨噬细胞衍生的产物递送至皮肤。