Sikorski E E, Gerberick G F, Ryan C A, Miller C M, Ridder G M
Human Safety Department, Procter & Gamble Company, Cincinnati, Ohio, USA.
Fundam Appl Toxicol. 1996 Nov;34(1):25-35. doi: 10.1006/faat.1996.0172.
The murine local lymph node assay (LLNA) measures in vivo proliferation in draining lymph nodes (DLN) following topical exposure to chemicals to assess contact sensitization potential. However, proliferation has also been observed with some irritants. To further characterize events in the DLN during the LLNA and distinguish allergens from irritants, phenotypic analysis of lymphocyte subsets was made following topical exposure. In preliminary studies, mice were treated on the ears for 3 consecutive days, and 48 hr following the final application, analysis of CD3, CD4, CD8, and B220 expression was evaluated by flow cytometry. The allergens oxazolone (OXAZ) and picryl chloride (TNCB) and the irritant benzalkonium chloride (BC) increased cell number compared to vehicle. The increase in lymph node cellularity for these materials was due to an increase in the total number of T and B lymphocytes. Interestingly, even though contact sensitization is a cell-mediated immune response (Th1), mice exposed to the contact allergens showed a preferential increase in B lymphocytes in the DLN as seen by an increase in the percentage of B220+ cells. The percentage of B220+ cells was 13.1 and 36.1% for OXA and TNCB, respectively, compared to percentages of 7.4 and 9.3% for irritant and vehicle, respectively. With some allergens, a concomitant decrease in the percentage of CD3+ cells was seen. Time course studies demonstrated the increase in the percentage of B220+ cells was seen in allergen treated mice by 24 hr after the final application of material, plateaued by 48 hr, and was still elevated by 96 hr. In allergen-treated mice, percentages of B220+ cells increased dose dependently. Further studies were performed to evaluate additional contact allergens and irritants and determine if evaluation of flow cytometric parameters could potentially identify contact allergens and differentiate them from irritants. Analysis of data from these studies, which examined a total of five contact allergens and six irritants, showed that the modifications to the LLNA improved the identification of irritants and allergens in individual experiments by including both phenotypic analysis of the DLN and cell number per node as endpoints rather than either endpoint alone.
小鼠局部淋巴结试验（LLNA）通过局部接触化学物质后测量引流淋巴结（DLN）中的体内增殖情况，以评估接触致敏潜力。然而，一些刺激物也会引起增殖。为了进一步描述LLNA过程中DLN内的事件，并区分过敏原和刺激物，在局部接触后对淋巴细胞亚群进行了表型分析。在初步研究中，连续3天对小鼠耳部进行处理，在最后一次给药后48小时，通过流式细胞术评估CD3、CD4、CD8和B220的表达。与赋形剂相比，过敏原恶唑酮（OXAZ）和苦味酸氯（TNCB）以及刺激剂苯扎氯铵（BC）使细胞数量增加。这些物质导致淋巴结细胞增多是由于T淋巴细胞和B淋巴细胞总数增加。有趣的是，尽管接触致敏是一种细胞介导的免疫反应（Th1），但接触过敏原的小鼠DLN中B淋巴细胞优先增加，表现为B220+细胞百分比增加。对于OXAZ和TNCB，B220+细胞的百分比分别为13.1%和36.1%，而刺激剂和赋形剂的百分比分别为7.4%和9.3%。对于一些过敏原，可观察到CD3+细胞百分比随之降低。时间进程研究表明，在最后一次给药后24小时，过敏原处理的小鼠中B220+细胞百分比增加，48小时达到平稳，96小时仍升高。在过敏原处理的小鼠中，B220+细胞百分比呈剂量依赖性增加。进行了进一步研究以评估更多的接触过敏原和刺激物，并确定流式细胞术参数评估是否有可能识别接触过敏原并将其与刺激物区分开来。这些研究共检测了5种接触过敏原和6种刺激物，对数据的分析表明，对LLNA的改进通过将DLN的表型分析和每个淋巴结的细胞数量作为终点，而非仅采用单一终点，提高了个体实验中对刺激物和过敏原的识别能力。
