Role of LAIR-1 and CD300 Family Receptors in Regulating Inflammation

我们在B6背景下建立了LAIR-1-/-小鼠，用于研究LAIR-1的体内功能。淋巴器官的表型分析表明，LAIR-1+/+和LAIR-1-/-动物之间没有太大的差异。我们观察到LAIR-1-/-小鼠的脾B细胞百分比略有增加，同时T细胞减少，主要是由于CD8T细胞的减少。这可能不是由于T淋巴细胞的异常运输所致，因为LAIR-1+/+和LAIR-1-/-T细胞同等地运输到外周淋巴器官。在肠道中，我们观察到LAIR-1-/-小鼠的T细胞略有增加，同时NKG2D表达增加。体外实验表明，OT-II LAIR-1-/-CD4T细胞与负载OT-II OVA多肽的APC共同培养时，其增殖能力低于OT-II LAIR-1+/+CD4T细胞。为了研究体内的免疫反应，我们用TNP-OVA免疫动物，发现LAIR-1-/-小鼠的类别转换受到影响。这些动物产生较低水平的IgG2a和IgG2b，而转换为IgG1不受影响。通过使用T细胞特异性LAIR-1-/-动物(CD4CRE LAIR-1FLOX/FLOX)，我们证实类别转换中的缺陷是T细胞特异性的。以前的研究人员已经发表了小鼠B细胞不表达LAIR-1；然而，最近我们发现边缘带B细胞表达LAIR-1。其他初步结果表明，在LAIR-1-/-小鼠中，在化学性腹膜炎模型中，巨噬细胞和嗜酸性粒细胞在腹膜中的募集发生了显著变化，这表明LAIR-1在这些细胞类型向炎症部位运输方面发挥了作用。 我们研究了保守的R65K突变对LAIR-1配体结合和功能的影响。与表达LAIR-1野生型(Wt)的细胞相比，LAIR-1 R65K细胞与胶原的结合显著减少，这与LAIR-1与胶原蛋白相互作用部位的极化水平降低有关。LAIR-1wt和R65K细胞在抗LAIR-1mAb作用下均能产生细胞内信号，但只有LAIR-1wt细胞对胶原蛋白或基质有反应。表面等离子体共振分析表明，与LAIR-1wt相比，LAIR-1R65K蛋白对I型胶原的亲和力显著降低。同样，LAIR-1R65K蛋白也降低了对表达跨膜型XVII胶原的细胞的亲和力。因此，单个残基Arg65对于LAIR-1与胶原蛋白的相互作用至关重要。

We have generated LAIR-1 -/- mice on a B6 background to study the in vivo function of LAIR-1. Phenotypic analysis of lymphoid organs did not show large differences between LAIR-1 +/+ and LAIR-1 -/- animals. We have observed a slight increase in the percentage of splenic B cells in the LAIR-1 -/- mice, along with a decrease in T cells, mostly because of a decrease in CD8 T cells. This probably does not result from abnormal trafficking of T lymphocytes, since LAIR-1 +/+ and LAIR-1 -/- T cells traffic equally to peripheral lymphoid organs. In the gut we have observed a slight increase of T cells in the LAIR-1 -/- mice, along with an increase in the NKG2D expression. In vitro experiments showed that OT-II LAIR-1-/- CD4 T cells proliferated less than the OT-II LAIR-1+/+ CD4 T cells when they are cultured with APC loaded with OT-II OVA peptide. To study the immune response in vivo, we have immunized the animals with TNP-OVA and found that class switching is affected in LAIR-1 -/- mice. These animals produced lower levels of IgG2a and IgG2b, while switching to IgG1 is not affected. By using T cell specific LAIR-1 -/- animals (CD4 Cre LAIR-1flox/flox), we confirmed that the defect in class switching is T cell specific. Previous investigators have published that mouse B cells do not express LAIR-1; however, recently we have found that marginal zone B cells are positive for LAIR-1 expression. Other preliminary results have shown that in the LAIR-1 -/- mice there are significant alterations in the recruitment of macrophages and eosinophils into the peritoneum in a model of chemical peritonitis, suggesting a role for LAIR-1 in the trafficking of these cell types towards sites of inflammation. We investigated the effect of a conservative R65K mutation on LAIR-1 ligand binding and function. Compared with LAIR-1 wild-type (wt)-expressing cells, LAIR-1 R65K cells show markedly reduced binding to collagen, which correlates with a reduced level of LAIR-1 polarization to the site of interaction with collagens. Both LAIR-1 wt and R65K cells can generate intracellular signals when ligated by anti-LAIR-1 mAb, but only LAIR-1 wt cells respond to collagens or matrigel. In agreement, surface plasmon resonance analyses showed that LAIR-1 R65K protein has markedly reduced avidity for collagen type I compared with LAIR-1 wt. Likewise, LAIR-1 R65K protein has decreased avidity for cells expressing transmembrane collagen XVII. Thus, a single residue, Arg65, is critical for the interaction of LAIR-1 with collagens.