Cytokine Dysregulation in GM-CSF Autoimmunity

描述（由申请人提供）：特发性肺泡蛋白沉积症（PAP）是一种新发现的自身免疫性疾病，其特征为粒细胞-巨噬细胞集落刺激因子（GMCSF）中和自身抗体和肺表面活性物质代谢严重失调，对GM-CSF缺陷小鼠的研究证明了正常表面活性物质稳态中需要GM-CSH，该小鼠发展为PAP样综合征，可通过外源性GM-CSF逆转。GM-CSF如何维持表面活性剂稳态尚不清楚。人PAP肺泡巨噬细胞（AM）可产生并应答GM-CSF，但其核转录因子过氧化物酶体增殖物激活受体γ（PPARgamma）的表达较低。GM-CSF上调PPARgamma，提示人PAP中存在GM-CSF-PPARgamma连接。有趣的是，在实验模型中，PPARgamma缺乏也与B细胞过度活跃和自身免疫有关。其他与B细胞功能障碍相关的细胞因子异常发生在PAP中。白细胞介素-10（IL-10）是一种刺激抗体合成的多效性细胞因子，在PAP肺和血液中升高，并阻断AM GM-CSF的产生。激活素A是一种可抑制B细胞周期的细胞因子，其被GM-CSF上调，并且在PAP中也被降低。因此，这些数据表明，PAP中存在抗GMCSF的中和性自身抗体导致细胞因子失调、严重受损的表面活性剂catalysts和临床疾病。B淋巴细胞的耗竭在自身免疫性疾病中显示出有希望的临床结果。利妥昔单抗是一种针对B细胞特异性膜抗原CD 20的鼠-人嵌合单克隆抗体，可选择性耗竭B细胞。基于我们的PAP数据，假设在利妥昔单抗临床试验的背景下减少PAP B细胞将降低抗GM-CSF抗体的水平，从而解决肺部疾病。将在基线和利妥昔单抗治疗后对来自肺和血液的免疫细胞进行目的1-3中概述的机制研究。目标1将调查：（a）IL-10、激活素A和PPAR γ对未处理的PAP B细胞增殖和抗GM-CSF合成的影响;和（B）B细胞水平和抗GM-CSF升。目的2将检查PAP AM在局部B细胞调节中的作用，具体地：（a）GM-CSF对IL-10、激活素A和卵泡抑素（激活素A抑制剂）表达的作用;和（B）激活素A对PPAR γ表达的作用。目的3将检查PAP T细胞的：（a）PAP B细胞合成抗GM-CSF的调节;和（B）调节表型（CD 4/CD 25、PPARgamma表达）、细胞因子（激活素A、IL-2、IL-10）和增殖。本申请的长期目标是描述PAP病理生理学中免疫细胞、细胞因子和抗GM-CSF自身抗体之间的关系。利妥昔单抗试验提供了一个独特的机会，探讨B细胞耗竭对PAP肺AM功能的影响，同时研究可能改善PAP肺部疾病的新疗法。

DESCRIPTION (provided by applicant): Idiopathic pulmonary alveolar proteinosis (PAP) is a newly recognized autoimmune disorder characterized by neutralizing autoantibodies to granulocyte-macrophage colony stimulating factor (GMCSF) and severely dysregulated lung surfactant metabolism, the GM-CSh requirement in normal surfactant homeostasis is evidenced by studies of GM-CSF-deficient mice which develop a PAP-like syndrome reversible with exogenous GM-CSF. How GM-CSF maintains surfactant homeostasis is unknown. Human PAP alveolar macrophages (AM) can produce and respond to GM-CSF but have low expression of the nuclear transcription factor, peroxisome proliferator-activated receptor qamma (PPARgamma). GM-CSF upregulates PPARgamma, suggesting a GM-CSF-PPARgamma link in human PAP. Interestingly, PPARgamma deficiency is also linked to B cell hyperactivity and autoimmunity in experimental models. Other cytokine anomalies associated with B cell dysfunction occur in PAP. lnterleukm-10 (IL-10), a pleiotropic cytokine that stimulates antibody synthesis, is elevated in PAP lung and blood and blocks AM GM-CSF production. Activin A, a cytokine that may restrain B cell cycling, is upregulated by GM-CSF, and is also reduced in PAP. Thus, these data suggest that the presence of neutralizing auto-antibodies to GMCSF in PAP results in cytokine dysregulation, severely impaired surfactant catabolism and clinical disease. Depletion of B lymphocytes has shown promising clinical results in autoimmune disease. Rituximab, a chimeric murine-human monoclonal antibody directed against the B cell-specific membrane antigen CD20, selectively depletes B cells. Based upon our PAP data, it is hypothesized that reducing PAP B Cells in the context of a Rituximab clinical trial will diminish levels of anti- GM-CSF antibodies, and thus resolve pulmonary disease. Mechanism studies outlined in Aims 1-3 will be conducted on immune cells from lung and blood at baseline and post Rituximab therapy. Aim 1 will investigate: (a) effects of IL-10, activin A, and PPARgamma on untreated PAP B cell proliferation and anti- GM-CSF synthesis; and (b) B cell levels and anti-GM-CSF liters. Aim 2 will examine the role of PAP AM in localized B-cell regulation, specifically: (a) GM-CSF effect on expression of IL-10, activin A and follistatin, (activin A inhibitor); and (b) activin A effect on PPARgamma expression. Aim 3 will examine PAP T cells for: (a) regulation of PAP B cell synthesis of anti-GM-CSF; and (b) regulatory phenotype (CD4/CD25, PPARgamma expression), cytokines (activin A, IL-2, IL-10), and proliferation. The long-term objective of this application is to delineate relationships among immune cells, cytokines and anti-GM-CSF autoantibody in PAP pathophysiology. The rituximab trial offers a unique opportunity to explore effects of B cell depletion on PAP pulmonary AM function and at the same time investigate new therapy that may improve the lung disease in PAP.