Role of the Orphan Receptors, LAIR-1,Toso and CD300, in Regulating Inflammation

为了研究LAIR-1的体内功能，我们在C57BL/6背景下建立了LAIR-1-/-和LAIR-1FLOX/FLOX小鼠。虽然LAIR-1-/-小鼠是健康的、多产的，在无病原体的环境中也有正常的寿命，但它们确实表现出不同于野生型(Wt)小鼠的某些表型特征。他们的脾B细胞和树突状细胞比例增加，而T细胞水平降低，其中更高比例的是CD4+CD25+。随着LAIR-1-/-小鼠年龄的增长，脾T细胞群表现出更高的激活T细胞频率和更高比例的效应/记忆T细胞。由于LAIR-1+/+和LAIR-1-/-T细胞对外周淋巴器官的转运能力相同，这可能不是T淋巴细胞异常转运的结果。功能实验表明，抗原特异性LAIR-1-/-CD4T细胞的增殖明显低于抗原特异性LAIR-1+/+CD4T细胞。尽管先前有报道称小鼠B细胞表达LAIR-1，但我们发现边缘带B细胞表达LAIR-1。LAIR-1-/-小鼠血清中的IgG3和IgG1水平较低，在TNP-OVA的T依赖免疫反应中，LAIR-1-/-小鼠转换为IgG2a和IgG2b的效率较低，而转换为IgG1的效果不受影响。采用实验性自身免疫性脑脊髓炎(EAE)和实验性结肠炎等多种疾病模型，观察了LAIR-1缺陷对小鼠体内免疫功能的影响，结果表明，LAIR-1-/-和LAIR-1+/+小鼠的免疫应答无明显差异。综上所述，这些观察表明，LAIR-1在塑造免疫反应方面发挥了作用，这种免疫反应可能在体内被其他抑制性受体补偿(提交和修改中)。 免疫调节受体CD300a在人B细胞上表达。幼稚B细胞表达这种受体的水平非常低，而记忆B细胞和浆母细胞/细胞表达不同水平的CD300a。生发中心B细胞CD300a表达阴性。通过BCR和TLR9刺激幼稚B细胞，以及T细胞的帮助，未能上调CD300a细胞表面的表达，尽管记忆标记CD27的表达增加，CD305的表达下调。TLR9单独刺激显著增加记忆B细胞CD300a的表达，而IL-4和转化生长因子-β1对记忆B细胞CD300a的表达起负调控作用。Bcr和CD300a共连接可抑制单独bcr连接引起的钙动员和NFAT转录活性。用siRNA抑制原代B细胞CD300a的表达，可促进BCR介导的增殖，从而证实CD300a的抑制能力。最后，我们发现HIV感染者循环B细胞中CD300a的表达水平下调。总之，这些数据显示了一种抑制B细胞活性的新机制，并表明CD300a在HIV诱导的免疫缺陷中观察到的B细胞功能障碍中可能起到作用。 据报道，CD300f在多发性硬化症小鼠模型中负向调节树突状细胞和T细胞之间的相互作用，并作为抗炎分子发挥作用。我们发现CD300f/Fc嵌合蛋白特异性地结合到凋亡/死亡的脾细胞，以及饥饿或辐射的淋巴细胞系中的凋亡细胞；这一观察延伸到昆虫细胞。CD300f还与PMA/离子霉素激活的脾细胞和Ag刺激的T细胞结合，这种相互作用被Annexin V抑制。通过含有磷脂的脂质体的ELISA法、共沉淀法和表面等离子共振实验，我们发现CD300f优先与磷脂酰丝氨酸结合，需要一个金属离子。CD300f在细胞系中的外源表达可增强细胞对凋亡细胞的吞噬功能。我们的结论是，CD300f的表达向髓系细胞传递了识别磷脂酰丝氨酸的额外能力。这种识别的结果可能会随着整体受体的定性和定量含量以及表达效应细胞的信号能力而变化，但吞噬功能增强是一个可测量的结果。 TOSO是NK细胞高表达的免疫球蛋白基因超家族成员。我们证实，该受体与IgM结合，并不作为抗细胞凋亡分子发挥作用。关于这种受体在NK细胞上的功能目前还不清楚。我们的结果表明，在原代NK细胞上交联该受体可导致胞浆内酪氨酸残基的磷酸化，并激活细胞内的激酶，表明该受体的连接能够激活NK细胞。我们还了解到，NK细胞暴露于IL-2可显著下调FcmuR的表达。由于激活的NK细胞分泌IL-2，这种下调可能是为了避免过度炎症，从而可能产生功能性后果。

To study the in vivo function of LAIR-1, we generated LAIR-1-/- and LAIR-1flox/flox mice on a C57Bl/6 background. Although LAIR-1-/- mice are healthy and fertile, and have normal longevity in pathogen free environment, they do show certain phenotypic characteristics distinct from wild-type (wt) mice. They develop an increased percentage of splenic B cells and dendritic cells, along with decreased levels of T cells, of which a higher frequency are CD4+CD25+. As the LAIR-1-/- mice age, the splenic T cell population shows a higher frequency of activated T cells and a higher percentage of effector/memory T cells. Since LAIR-1+/+ and LAIR-1-/- T cells traffic with equal proficency to peripheral lymphoid organs, this is probably not the result of abnormal T lymphocyte trafficking. Functional experiments showed that antigen (Ag) specific LAIR-1-/- CD4 T cells proliferate significantly less than Ag specific LAIR-1+/+ CD4 T cells. Although mouse B cells have previously been reported to be negative for LAIR-1 expression, we find that marginal zone B cells express LAIR-1. LAIR-1-/- mice have lower levels of IgG3 and IgG1 in serum and, in response to T-dependent immunization with TNP-OVA, LAIR-1-/- switch less efficiently to IgG2a and IgG2b production, while switching to IgG1 is not affected. Several mouse disease models, including those for experimental autoimmune encephalomyelitis (EAE) and experimental colitis, were utilized to examine the effect of LAIR-1 deficiency in vivo, and no difference in the response of LAIR-1-/- and LAIR-1+/+ mice were observed. Taken together, these observations indicate that LAIR-1 plays a role in shaping the immune response that may be compensated for in vivo by other inhibitory receptors(submitted and in revision). The immunomodulatory receptor CD300a is expressed on human B cells. Nave B cells express very low levels of this receptor, while memory B cells and plasma blasts/cells express variable levels of CD300a. Germinal center B cells are negative for CD300a expression. Stimulation of naive B cells via BCR and TLR9, along with T cell help, failed to up-regulate CD300a cell surface expression despite the increased expression of the memory marker CD27 and the down-regulation of CD305. TLR9 stimulation alone significantly increased CD300a expression on memory B cells, whereas IL-4 and TGF-beta1 act as negative regulators of CD300a expression on memory B cells. Co-ligation of BCR and CD300a inhibits Ca2+ mobilization and NFAT transcriptional activity evoked by BCR ligation alone. Suppression of CD300a expression by primary B cells with siRNA resulted in increased BCR-mediated proliferation, thereby confirming the inhibitory capacity of CD300a. Finally, we show that of CD300a expression levels are down-regulated in the circulating B cells of HIV-infected patients. Altogether, these data demonstrate a novel mechanism for suppressing the activity of B cells and suggest a potential role for CD300a in the B cell dysfunction observed in HIV induced immunodeficiency. Reportedly, CD300f negatively regulates interactions between dendritic and T cells, and acts as an anti-inflammatory molecule in a multiple sclerosis mouse model. We found that a CD300f/Fc chimeric protein specifically binds to apoptotic/dead splenocytes and to apoptotic cells from starved or irradiated lymphocytic cell lines; an observation extended to insect cells. CD300f also binds PMA/ionomycin-activated splenocytes and Ag-stimulated T cells, an interaction inhibited by Annexin V. By ELISA, co-sedimentation and surface plasmon resonance using phospholipid containing liposomes, we show that CD300f preferentially binds phosphatidylserine and requires a metal ion. Exogenous expression of CD300f in cell lines results in enhanced phagocytosis of apoptotic cells. We conclude that expression of CD300f conveys additional capacity to recognize phosphatidylserine to myeloid cells. The result of this recognition may vary with the overall qualitative and quantitative receptor content, as well as signaling capacity of the expressing effector cell, but enhanced phagocytosis is one measurable outcome. Toso is a member of immunoglobulin gene superfamily that is highly expressed on NK cells. We cofirmed that this receptor binds IgM and does not function as an anti-apoptotic molecule. Nothing is known about the function of this receptor on NK cells. Our results show that crosslinking this receptor on primary NK cells leads to phosphorylation of cytoplasmic tyrosine residues, as well as to the activation of intracellular kinases, indicating that ligation of this receptor is capable of activating NK cells. We have also learned that exposure of NK cells to IL-2 markedly down-regulates the expression of FcmuR. Since activated NK cells secrete IL-2, this down-modulation may occur in order to avoid excess inflammation and thus may have functional consequences.