THE ROLE OF UROKINASE IN LUNG T LYMPHOCYTE RESPONSES

我们已经证明，uPA是体内有效的肺防御新生隐球菌(C.neo)感染所必需的。宿主对C.neo的防御完全依赖于T细胞。在没有uPA的情况下，肺中的CD4细胞无法对感染做出反应，而uPA阴性/阴性的动物死于播散性隐球菌病。我们发现，在没有uPA的情况下，T细胞损伤并不是C.neo所特有的。在体内，uPA阴性/阴性小鼠不能增加肺内CD4细胞对卡氏肺孢子虫的反应，也不能清除感染。我们发现，在缺乏uPA的情况下，T细胞的增殖反应严重受损。在体外，uPA阴性/阴性淋巴细胞不会经历抗原特异性的增殖，也不会对CD3交联或有丝分裂原产生的TCR介导的信号做出反应而增殖。在体内对T细胞特异性病原体的有效宿主防御；以及体外由抗原、有丝分裂原或CD3交联物诱导的正常反应T细胞增殖试验，主要通过TCR-蛋白激酶级联介导的共同信号通路连接，并与CD28和CD25传递的共刺激信号相耦合。我们的假设是，在缺乏uPA的情况下，T淋巴细胞通过JAK/STAT的细胞内信号转导减弱，导致细胞因子产生不足，细胞因子反应性减弱。缺乏JAK/STAT信号导致T细胞活化和增殖受阻；T细胞对TCR介导的进一步信号无反应。1.定位T细胞信号转导通路中的生化阻断(S)。A)确定uPA的缺失是否会削弱TCR介导的信号转导。B)确定uPA的缺失是否会减弱CD28和CD25介导的信号转导。C)确定uPA是否激活小鼠T淋巴细胞上的JAK/STAT信号。2.确定T细胞信号异常的功能后果。A)根据激活抗原的表达、细胞因子的产生和对IL-12的反应性，确定淋巴细胞激活时对uPA的需求。B)确定uPA缺失/缺失淋巴细胞信号的缺陷是否导致无能、细胞周期停滞或细胞凋亡。3.体外检测T细胞活化和增殖过程中uPA和uPAR的表达和转运。确定重组uPA能否逆转uPA阴性/阴性淋巴细胞的功能异常。

We have demonstrated that uPA is required for effective pulmonary defense against Cryptococcus neoformans (C. neo) infection in vivo. Host defense against C. neo is absolutely T cell dependent. In the absence of uPA, lung accumulation of CD4 cells fails to occur in response to infection, and the uPA minus/minus animals die from disseminated Cryptococcosis. We found that the T cell impairment in the absence of uPA is not specific to C. neo. In vivo, uPA minus/minus mice fail to increase pulmonary CD4 cells in response to P. carinii, and do not clear the infection. We have found that T cell proliferative responses are profoundly impaired in the absence of uPA. In vitro, uPA minus/minus lymphocytes do not undergo antigen-specific proliferation, nor do they proliferate in response to TCR- mediated signals generated by CD3-cross-linking, or by mitogen. Effective host defense to T-cell specific pathogens in vivo; and normal responses in vitro T cell proliferation assays induced by antigen, mitogen, or by CD3-cross-linking, are linked by a common signaling pathway mediated primarily through the TCR-protein kinase cascade, and coupled to co-stimulatory signals delivered by CD28 and CD25. It is our hypothesis that in the absence of uPA, T lymphocyte intracellular signaling via Jak/STAT is diminished leading to inadequate cytokine production and diminished cytokine responsiveness. The lack of Jak/STAT signaling results in blocked T cell activation and proliferation; and T cell unresponsiveness to further TCR-mediated signaling. 1. Localize the biochemical block(s) in T cell signaling cascades. a) Determine if the absence of uPA diminishes TCR- mediated signaling. b) Determine if the absence of uPA diminishes CD28- and CD25-mediated signaling. c) Determine whether uPA activates Jak/STAT signaling in murine T lymphocytes. 2. Determine the functional consequences of aberrant T cell signaling. a) Determine the requirement for uPA in lymphocyte activation as measured by expression of activation antigens, elaboration of cytokines, and responsiveness to IL-12. b) Determine whether the defects in uPA minus/minus lymphocyte signaling result in anergy, cell cycle arrest, or apoptosis. 3. Determine the cellular expression and trafficking of uPA and uPAR during T cell activation and proliferation in vitro. Determine if recombinant uPA can reverse the functional abnormalities in uPA minus/minus lymphocytes.