MDDCs exhibit high lysosomal protease activity <i>in vitro</i> compared to CD34DCs.

(A) Cell lysates prepared from cultures of MΦs, MDDCs, and CD34DCs were incubated together with OVA in either degradation reaction buffer (pH 4.5) or control buffer (pH 7.4). A sample containing OVA in reaction buffer with no lysate was loaded in the first lane as a non-degraded sample. Partial degradation of OVA by MΦs and MDDCs is evident while no degradation by CD34DCs is seen. (B) Quantitation of the rate of degradation by these cells using a self-quenching fluorescent protein substrate demonstrates that immature (i-) MDDCs are equivalent to MΦs in proteolytic capacity, mature (m-) MDDCs are 2-fold less proteolytic than MΦs, while i-CD34DCs and m-CD34DCs are 17- and 28-fold less proteolytic than MΦs, respectively. (C) Cell lysates were prepared from monocyte cultures as they differentiated into either MDDCs or MΦs and were analyzed by immunoblot for catB. CatB expression in MDDCs culminates at day 4 and is diminished following maturation on day 5 and analysis on day 6. MΦs exhibit a steady increase in catB expression from a low level at day 2 to a high level at day 6. (D) Cell lysates of culture samples from (C) were assessed for degradative capacity by incubation with OVA in either reaction buffer (pH 4.5) or control buffer (pH 7.4). Degradation at pH 4.5 correlates with protease expression levels. Relative fluorescence units (RFU).(A) 将从巨噬细胞(MΦs)、髓样树突状细胞(MDDCs)以及CD34DCs培养物中制备的细胞裂解液，与卵清蛋白(OVA)共同置于pH 4.5的降解反应缓冲液或pH 7.4的对照缓冲液中孵育。第一泳道加载仅含OVA的反应缓冲液样本，作为未降解对照。实验结果可见，MΦs与MDDCs可对OVA产生部分降解，而CD34DCs未表现出明显的降解活性。 (B) 采用自淬灭荧光蛋白底物对上述细胞的降解速率进行定量分析，结果显示：未成熟型(i-)MDDCs的蛋白水解能力与MΦs相当；成熟型(m-)MDDCs的蛋白水解能力较MΦs低2倍；而未成熟型(i-)CD34DCs与成熟型(m-)CD34DCs的蛋白水解能力分别较MΦs低17倍与28倍。 (C) 收集单核细胞向MDDCs或MΦs分化过程中的培养样本，制备细胞裂解液后通过免疫印迹(immunoblot)检测组织蛋白酶B(catB)的表达水平。MDDCs中catB的表达于第4天达到峰值，在第5天诱导成熟后，于第6天的检测样本中表达量显著下降。而MΦs中catB的表达则从第2天的低水平逐步升高，至第6天达到最高表达量。 (D) 对(C)中获取的培养样本的细胞裂解液，通过将其与OVA共同置于pH 4.5的降解反应缓冲液或pH 7.4的对照缓冲液中孵育，以评估其蛋白降解能力。pH 4.5条件下的降解活性与样本中的蛋白酶表达水平呈正相关。相对荧光单位(RFU)。