Mechanisms of Leukocyte Migration Following Cytokine Administration to Mice

在不同的器官微环境中，不同的白细胞亚群和基质细胞之间的相互作用对于优化器官特异性免疫反应至关重要，从而限制转移形成和控制疾病诱导的炎症。IL-12和IL-18是对自然杀伤(NK)、NKT和T细胞有很强免疫调节作用的细胞因子，对不同器官中的NKT/NK亚群有非常不同的影响。肝脏是转移瘤形成的主要靶点。我们利用小鼠肝转移模型研究了IL-18和/或IL-12细胞因子的抗肿瘤活性以及NK细胞和NKT细胞在这些反应中的作用。与单独使用两种细胞因子相比，使用IL-18和IL-12治疗的小鼠显著减少了肝脏中肿瘤结节的数量。正如预期的那样，IL-18和IL-12在荷瘤小鼠体内协同刺激全身干扰素-γ的增加。IL-18和IL-12联合治疗在干扰素-γ(-/-)小鼠体内的抗肿瘤活性被取消。通过细胞内染色，NK和NKT细胞被鉴定为IL-18和/或IL-12处理的小鼠肝脏中产生干扰素-γ的主要细胞。IL-18和IL-12联合治疗可使小鼠肝脏NK细胞增加，而肝脏NKT细胞减少。用抗asGM1抗体优先去除NK细胞后，IL-18和IL-12联合治疗的抗肿瘤活性部分丧失，提示NK细胞是肿瘤消退机制的重要组成部分。相比之下，使用BetaGalCer优先清除NKT细胞减少了赋形剂对照组或单独使用IL-18治疗的小鼠的肝肿瘤结节数量。同样，所有治疗方法都显示出CD1d(-/-)小鼠的抗肿瘤活性增加，因为CD1d(-/-)小鼠缺乏NKT细胞。因此，我们的数据表明，IL-18和IL-12诱导的小鼠抗肿瘤活性是依赖于NK和干扰素-γ的，并且能够克服NKT细胞的内源性免疫抑制效应。因此，这些结果表明，在优先消耗NKT细胞的同时增强NK细胞数量和功能的免疫治疗方法在治疗肝癌方面可能是有效的。由于IL-18联合IL-12治疗荷瘤小鼠在可检测到的不变NKT(INKT)细胞数量减少的同时，产生了显著的抗肿瘤活性，因此我们比较了α-GalCer和IL-18+IL-12的iNKT细胞调节机制。我们发现，IL-18、IL-12急性处理导致iNKT细胞亚群NK1.1()丢失，而aGalCer急性处理在24小时耗尽所有肝脏iNKT细胞。然而，其他淋巴组织对这些细胞的丢失知之甚少，这表明肝脏微环境是iNKT细胞的关键调节因素。72小时后，经α-GalCer和IL-18+IL-12处理后，iNKT细胞在肝脏中重新增殖和扩增。为了研究这种扩张，我们在用IL-18加IL-12或aGalCer急性治疗24小时后给小鼠注射BrdU，发现有高比例的iNKT细胞增殖。我们进一步发现，iNKT在aGalCer后的再分化和扩张需要脾，而IL-18和IL-12联合治疗则不需要，因为在去脾的小鼠中没有观察到扩张。接下来，我们研究了用aGalCer或IL-18加IL-12慢性治疗的小鼠，发现aGalCer导致肝脏iNKT细胞严重丢失，而只有IL-18加IL-12导致全身iNKT细胞丢失。在两种治疗后，需要长期(60天)胸腺发育来重新填充肝脏。这些数据揭示了肝脏微环境在调节iNKT细胞对炎症信号的反应中的重要作用。

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.