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C57 B1/6背景下产生了LAIR-1-/-和LAIR-1flox/flox小鼠。虽然LAIR-1-/-小鼠是健康的和可生育的，并且在无病原体的环境中具有正常的寿命，但是它们确实显示出与野生型（wt）小鼠不同的某些表型特征。它们产生脾B细胞和树突状细胞的百分比增加，沿着T细胞水平降低，其中较高频率是CD 4 + CD 25+。随着LAIR-1-/-小鼠年龄的增长，脾T细胞群显示出更高频率的活化T细胞和更高百分比的效应/记忆T细胞。由于LAIR-1+/+和LAIR-1-/- T细胞以相同的效率运输到外周淋巴器官，这可能不是异常T淋巴细胞运输的结果。功能实验显示抗原（Ag）特异性LAIR-1-/-CD 4 T细胞增殖显著低于Ag特异性LAIR-1+/+ CD 4 T细胞。虽然小鼠B细胞先前已报道为LAIR-1表达阴性，但我们发现边缘区B细胞表达LAIR-1。LAIR-1-/-小鼠在血清中具有较低水平的IgG 3和IgG 1，并且响应于TNP-0 VA的T依赖性免疫，LAIR-1-/-转换为IgG 2a和IgG 2b产生的效率较低，而转换为IgG 1不受影响。利用几种小鼠疾病模型，包括实验性自身免疫性脑脊髓炎（EAE）和实验性结肠炎的那些，来检查LAIR-1缺陷在体内的作用，并且没有观察到LAIR-1-/-和LAIR-1+/+小鼠的应答的差异。总之，这些观察结果表明，LAIR-1在塑造免疫应答中起作用，该免疫应答可以在体内由其他抑制性受体补偿（提交和修订中）。 免疫调节受体CD 300 a在人B细胞上表达。裸B细胞表达非常低水平的该受体，而记忆B细胞和血浆母细胞/细胞表达可变水平的CD 300 a。老年中心B细胞CD 300 a表达阴性。通过BCR和TLR 9刺激幼稚B细胞，沿着T细胞的帮助，尽管记忆标记物CD 27的表达增加和CD 305的下调，但不能上调CD 300 a细胞表面表达。TLR 9单独刺激显著增加记忆B细胞上的CD 300 a表达，而IL-4和TGF-β 1作为记忆B细胞上的CD 300 a表达的负调节剂。BCR和CD 300 a的共连接抑制单独BCR连接引起的Ca 2+动员和NFAT转录活性。用siRNA抑制原代B细胞的CD 300 a表达导致BCR介导的增殖增加，从而证实了CD 300 a的抑制能力。最后，我们发现，CD 300 a的表达水平下调，在循环B细胞的HIV感染患者。总之，这些数据证明了一种抑制B细胞活性的新机制，并表明CD 300 a在HIV诱导的免疫缺陷中观察到的B细胞功能障碍中的潜在作用。 据报道，CD 300 f负调节树突状细胞和T细胞之间的相互作用，并在多发性硬化症小鼠模型中充当抗炎分子。我们发现CD 300 f/Fc嵌合蛋白特异性结合凋亡/死亡脾细胞和来自饥饿或辐射淋巴细胞系的凋亡细胞;观察扩展到昆虫细胞。 CD 300 f还结合PMA/离子霉素激活的脾细胞和Ag刺激的T细胞，抑制膜联蛋白V的相互作用。通过ELISA，共沉降和表面等离子体共振使用含磷脂的脂质体，我们表明，CD 300 f优先结合磷脂酰丝氨酸，需要金属离子。CD 300 f在细胞系中的外源性表达导致凋亡细胞的吞噬作用增强。我们的结论是表达CD 300 f传达了额外的能力，以识别磷脂酰丝氨酸的骨髓细胞。这种识别的结果可能随总体定性和定量受体含量以及表达效应细胞的信号传导能力而变化，但增强的吞噬作用是一种可测量的结果。 Toso是免疫球蛋白基因超家族的成员，在NK细胞上高度表达。我们证实，这种受体结合IgM，并不作为一个抗凋亡分子。 目前还不清楚这种受体在NK细胞上的功能。我们的研究结果表明，交联这种受体上的初级NK细胞导致细胞质酪氨酸残基的磷酸化，以及细胞内激酶的激活，表明这种受体的连接是能够激活NK细胞。 我们还了解到NK细胞暴露于IL-2显著下调FcmuR的表达。由于活化的NK细胞分泌IL-2，这种下调可能发生，以避免过度炎症，因此可能具有功能性后果。

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.