Molecular analyzes of sensitization phase of contact hypersensitivity

本研究旨在阐明接触敏感性敏感期的分子机制，尤其是真皮巨噬细胞进入引流淋巴结以及致敏阶段淋巴结内细胞的生物学功能。(1)真皮巨噬细胞进入引流淋巴结：研究凝集素-糖类相互作用在致敏阶段表达巨噬细胞半乳糖C型凝集素(MGL1)的巨噬细胞进入中的作用。免疫组织化学显示，重组MGL1在LNS中的结合部位与致敏过程中表达MGL1的细胞明显从真皮迁移的分布相似。用固定化重组MGL1亲和层析成功地纯化了一个优势组分，经鉴定为唾液粘附素。固定化唾液酸粘附素实际上结合了重组的MGL1和MGL1转基因的CHO细胞。这种结合依赖于唾液酸粘附素上表达的碳水化合物。唾液酸粘附素和MGL1配体的免疫组织化学定位一致。此外，唾液粘附素和MGL1紧密分布在被膜下窦内。(2)致敏期淋巴结内真皮巨噬细胞的生物学功能：本研究的目的是探讨MGL1细胞对致敏期的调节作用。检测MGL细胞环氧合酶(COXJ-2)基因的表达。这些细胞表达COX-2mRNA，并释放COX-2依赖的前列腺素的最终产物PGE2。致敏阶段给予COX-2抑制剂NS-398，可抑制致敏小鼠淋巴结中PGE2的产生，并抑制致敏小鼠的抗原依赖性耳肿胀。

The aim of this proposal was to elucidate molecular mechanisms of sensitization phase of contact sensitivity, especially focusing on the entry of dermal macrophages to the draining lymph node and on the biological function of the cells inside the lymph node during the sensitization phase.(1) Entry of dermal macrophages to the draining lymph node : Involvement of lectin-carbohydrate interaction in the entry of macrophages expressing macrophage galactose-type C-type lectin (MGL1) in the sensitization phase was investigated. Immunohistochemical localization of binding sites of recombinant MGL1 in LNs was revealed to be similar to distribution of cells expressing MGL1 apparently migrated from the dermis during sensitization. Affinity chromatography with immobilized recombinant MGL1 successfully purified a predominant component that was identified as sialoadhesin. Immobilize sialoadhesin actually bound recombinant MGL1 as well as MGL1-transfected CHO cells. The binding was dependent on the carbohydrates expressed on sialoadhesin. Immunohistochemical localization of sialoadhesin and MGL1 ligand coincided. Furthermore, sialoadhesin and MGL1 closely distributed in subcapsular sinus.(2) Biological function of the dermal macrophages inside the lymph node during the sensitization phase : The aim of the study is to investigate how the MGL1 + cells modulate the sensitization phase. MGL+ cells were tested for the expression of cyclooxygenase (COXJ-2 mRNA. The cells expressed COX-2 mRNA and released PGE2, a final product of the COX-2-dependent prostanoids. The administration of a COX-2 inhibitor NS-398 during the sensitization phase inhibited the production of PGE2 in the lymph node and also antigen-dependent ear swelling of the sensitizad mice.