Mechanisms of Leukocyte Migration Following Cytokine Administration to Mice

不同器官白细胞亚群与基质细胞的相互作用 微环境对于优化器官特异性免疫反应至关重要， 转移形成并控制疾病引起的炎症。IL-12和IL-18是有效的 自然杀伤细胞（NK），NKT和T细胞的免疫调节细胞因子，并具有非常 对不同器官NKT/NK亚群的影响不同。肝脏是一个主要的目标， 转移瘤的形成。我们使用小鼠肝转移模型来研究抗肿瘤 IL-18和/或IL-12细胞因子的活性以及NK和NKT细胞对这些应答的作用。 用IL-18加IL-12治疗小鼠显著减少了肿瘤结节的数量。 肝脏中的作用比单独的细胞因子中的任一种更大。正如预期，IL-18加 IL-12刺激荷瘤小鼠中全身IFN-γ的协同增加。的 IL-18加IL-12治疗的抗肿瘤活性在IFN-γ（-/-）小鼠中被消除。使用 细胞内染色，NK和NKT细胞被鉴定为IFN-γ的主要产生者 在用IL-18和/或IL-12处理的小鼠的肝脏中。肝脏NK细胞增加， 每天用IL-18加IL-12处理小鼠，而肝NKT细胞通过这种方法减少 治疗用抗-asGM 1优先耗竭NK细胞导致部分丧失 IL-18加IL-12治疗的抗肿瘤活性，并揭示NK细胞是一种重要的 肿瘤消退机制的组成部分。与此相反， 具有β GalCer的NKT细胞减少了用β GalCer处理的小鼠中的肝肿瘤结节的数量。 载体对照或单独的IL-18。同样，所有治疗方法均显示 在缺乏NKT细胞的CD 1d（-/-）小鼠中的抗肿瘤活性。因此，我们的数据显示， 小鼠中IL-18加IL-12诱导的抗肿瘤活性是NK和IFN-γ依赖性的， 能够克服NKT细胞的内源性免疫抑制作用。因此，这些结果 这表明，免疫途径增强NK细胞数量和功能， 优先消耗NKT细胞可有效治疗肝癌。 因为IL-18加IL-12治疗荷瘤小鼠引起显著的抗肿瘤作用， 活性与不变NKT（iNKT）细胞的可检测数量减少同时，我们 比较了已知可调节iNKT的糖脂α-GalCer的iNKT细胞调节机制 细胞和IL-18加IL-12。我们发现用IL-18 + IL-12急性治疗诱导了 NK1.1（+）亚群的iNKT细胞，而用aGalCer的急性治疗耗尽了所有肝脏iNKT 细胞24小时然而，其他淋巴组织几乎不知道这些细胞的损失 提示肝脏微环境是iNKT细胞的关键调节因子。在72小时iNKT 在aGalCer和IL-18加IL-12处理后，细胞在肝脏中重新增殖和扩增 对小鼠为了研究这种扩展，我们在急性治疗后24小时向小鼠施用BrdU 用IL-18加IL-12或aGalCer处理，发现高百分比的增殖iNKT细胞。我们 进一步发现脾是iNKT再增殖和aGalCer后扩增所需的，但 而IL-18加IL-12处理则没有，因为在脾切除小鼠中没有观察到扩增。我们 接下来检查了用aGalCer或IL-18加IL-12对小鼠的长期治疗，发现aGalCer 诱导肝脏iNKT细胞的严重损失，而仅IL-18加IL-12引起全身损失 iNKT细胞需要长期（60天）的胸腺发育来重新填充 两种治疗后的肝脏。这些数据揭示了肝脏的重要作用 在调节iNKT细胞对炎症信号的反应中的作用。

Interaction between different leukocyte subsets and stromal cells in different organ microenvironments is critical for optimizing organ-specific immune responses that could limit metastasis formation and control disease-induced inflammation. IL-12 and IL-18 are potent immunoregulatory cytokines for natural killer (NK), NKT, and T cells, and have very distinctive effects on the NKT/NK subsets in different organs. The liver is a major target for the formation of metastases. We used a mouse model of liver metastasis to study the anti-tumor activity of IL-18 and/or IL-12 cytokines and the roles of NK and NKT cells to these responses. Treating mice with IL-18 plus IL-12 significantly reduced the number of tumor nodules in the liver to a greater degree than did either of the cytokines alone. As expected, IL-18 plus IL-12 stimulated a synergistic increase in systemic IFN-gamma in tumor bearing mice. The anti-tumor activity of IL-18 plus IL-12 therapy was abolished in IFN-gamma(-/-) mice. Using intracellular staining, NK and NKT cells were identified as the major producers of IFN-gamma in the livers of mice treated with IL-18 and/or IL-12. Liver NK cells were increased with daily treatment of mice with IL-18 plus IL-12 whereas liver NKT cells were diminished by this treatment. Preferential depletion of NK cells with anti-asGM1 resulted in a partial loss of the anti-tumor activity of IL-18 plus IL-12 therapy and revealed NK cells to be an important component of the mechanism for tumor regression. In contrast, the preferential depletion of NKT cells with betaGalCer decreased the number of liver tumor nodules in mice treated with vehicle control or IL-18 alone. Similarly, all treatment approaches showed increased anti-tumor activity in CD1d(-/-) mice, which lack NKT cells. Our data therefore shows that the IL-18 plus IL-12 induced anti-tumor activity in mice is NK and IFN-gamma dependent, and is able to overcome an endogenous immunosuppressive effect of NKT cells. These results thereby suggest that immunotherapeutic approaches that enhance NK cell numbers and function while preferentially depleting NKT cells could be effective in the treatment of cancer in the liver. Because IL-18 plus IL-12 treatment of tumor bearing mice elicited significant anti-tumor activity concurrent with a decrease in the detectable number of invariant NKT (iNKT) cells, we compared iNKT cell modulation mechanisms of alpha-GalCer, a glycolipid known to modulate iNKT cells, and IL-18 plus IL-12. We found acute treatment with IL-18 + IL-12 induced a loss of NK1.1(+) subset of iNKT cells, while acute treatment with aGalCer depleted all liver iNKT cells at 24hrs. However, other lymphoid tissues showed little to know loss of these cells suggesting the liver microenviroment is a critical regulator of iNKT cells. At 72hrs iNKT cells repopulated and expanded in the liver following aGalCer and IL-18 plus IL-12 treatment of mice. To study this expansion, we administered BrdU to mice 24hrs after acute treatment with IL-18 plus IL-12 or aGalCer and found a high percentage of proliferating iNKT cells. We further found the spleen was needed for iNKT repopulation and expansion following aGalCer, but not for IL-18 plus IL-12 treatment, as no expansion was observed in splenectomized mice. We next examined chronic treatment of mice with aGalCer or IL-18 plus IL-12 and found aGalCer induced a severe loss of liver iNKT cells, while only IL-18 plus IL-12 caused a systemic loss of iNKT cells. Long term (>60 days) thymic development was needed to repopulate the liver following both treatments. These data reveal an important role for the liver microenvironment in regulating iNKT cell response to inflammatory signals.