Phosphodiesterases as Therapeutic Targets: Sarcoidosis

临床研究：抑制与肉芽肿的形成和维持有关的炎性细胞因子的非类固醇药物在结节病的治疗中将是非常有用的治疗方法。在这方面，己酮可可碱(POF)已被报道抑制IL-2、IL-12、TNF-α的产生，并在一项开放标签的临床研究中，改善或稳定肺结节病。因此，我们进行了一项随机、双盲、安慰剂对照试验，以评估POF治疗是否能改善或稳定肺结节病，并作为皮质类固醇治疗的辅助手段是有益的。在我们的研究中，POF没有改善肺结节病患者的主要或次要终点。虽然肺结节病的复发不被认为是一个正式的终点，但接受POF治疗的患者中有5/13经历了疾病复发，而接受安慰剂治疗的患者中有12/14经历了疾病复发(P&lt；0.02)。安慰剂组没有自发缓解。24周后，经POF治疗的患者的平均泼尼松剂量显著低于安慰剂对照组患者(P&lt；0.006)。在接受POF治疗的患者中，有11/13出现了不良反应，主要是胃肠道反应，但没有接受安慰剂治疗的患者。因此，尽管POF在肺结节病中可能有节省皮质类固醇的作用，但在本研究中使用的药物浓度，胃肠道副作用是常见的报道。 基础科学研究：己酮可可碱(POF)是一种非特异性的磷酸二酯酶抑制剂，通过其已知的抑制肿瘤坏死因子-α(TNF-α)和IL-12等细胞因子的产生，可能对结节病患者有有益的影响。将正常人外周血单核细胞来源的巨噬细胞与大肠杆菌0128：B12脂多糖(LPS)(1ug/mL)和1 mM POF孵育18小时，然后提取RNA，与基于寡核苷酸的芯片杂交。在被脂多糖刺激至少2倍以上并被POF显著抑制的mRNAs中，有：干扰素刺激蛋白15 kDa(ISG15)、上皮膜蛋白3(EMP3)、2？-5？-寡腺苷合成酶3,100 kDa(OAS3)、骨髓基质细胞抗原2(BST2)和IPA蛋白酶体(Proome，巨痛)亚单位，β-9型(PSMB9)。特别是，内毒素使ISG15的表达增加8.2倍，而POF组的ISG15值仅为对照组的4.4倍(P&lt；0.005)。实时荧光定量聚合酶链式反应证实POF部分逆转了脂多糖对ISG15表达的刺激作用。ISG15可能是结节病的相关靶点，因为已知干扰素-α诱导ISG15会导致干扰素-α增加，这是一种与肉芽肿反应相关的关键Th1细胞因子。有趣的是，在正常志愿者的肺泡巨噬细胞中，内毒素未能诱导ISG15的表达，这表明ISG15在肺泡巨噬细胞和单核细胞来源的巨噬细胞中的表达可能受到不同的调控。 在另一项研究中，我们发现cAMP升高剂前列腺素E1和霍乱毒素(它们通过不同的机制激活腺酰环化酶)，以及环核苷酸磷酸二酯酶(PDE)抑制剂西洛胺(PDE3抑制剂)和罗利普兰(PDE4抑制剂)诱导FDCP2早幼粒细胞凋亡。CAMP类似物8-溴-环AMP和Sp-cAMP也可诱导这些细胞的凋亡。PKA抑制剂8-溴-RP-cAMP或胰岛素样生长因子-1(可同时激活FDCP2细胞中的PDE3和PDE4)可阻止cAMP提高剂诱导的细胞凋亡。在凋亡细胞中，促凋亡蛋白Bax从胞浆重新分布到细胞膜。这种重新分布可以通过用IGF-1处理细胞来防止。去掉培养上清液中的血清可导致FDCP2细胞中ERK1/2和PKB的去磷酸化，cAMP提高剂显著加速这一过程。在ERK1/2去磷酸化中，磷酸酶的激活比上游蛋白激酶的抑制更重要。我们的结果表明，在FDCP2细胞中，cAMP提高剂通过PKA和PP2A或PP2A样蛋白磷酸酶作用，抑制Raf-MEK-Erk和PKB信号，从而取消Erk和PKB介导的生存信号，同时Bax转位到膜上，可能导致Bax介导的细胞凋亡。另一方面，在这些细胞中，IGF-1促进抗凋亡或生存信号，很可能是通过两种机制。IGF-1通过依赖磷脂酰肌醇-3激酶(PI3-K)的途径激活ERK和PKB，从而促进包括PDE3和PDE4在内的抗凋亡信号，导致cAMP减少，从而抑制cAMP的抗增殖和促凋亡作用。综上所述，这些研究支持这样的假设，即在FDCP2细胞中，PDE起抗凋亡作用，而蛋白磷酸酶起促凋亡调节作用。

Clinical studies: Steroid-sparing drugs which suppress inflammatory cytokines that are associated with the formation and maintenance of granulomas would be very useful therapeutics in management of sarcoidosis. In this regard, pentoxifylline (POF) has been reported to inhibit production of IL-2, IL-12, TNF-alpha and, in an open-label clinical study, to improve or stabilize pulmonary sarcoidosis. We therefore conducted a randomized, double-blinded, placebo-controlled trial to assess whether POF treatment would improve or stabilize pulmonary sarcoidosis, and be beneficial as an adjunct to corticosteroid therapy. In our study, POF did not improve primary or secondary endpoints in patients with pulmonary sarcoidosis. Although recurrence of pulmonary sarcoidosis was not considered a formal endpoint, 5/13 POF-treated patients experienced disease recurrence versus 12/14 placebo-treated patients (P < 0.02). There were no spontaneous remissions in the placebo group. After 24 weeks, the mean prednisone dose for POF-treated patients was significantly lower than for placebo-controlled patients (P < 0.006). Adverse effects, primarily gastrointestinal, were reported in 11/13 POF-treated , but in no placebo-treated patients. Thus, although POF may have a corticosteroid-sparing effect in pulmonary sarcoidosis, with concentrations of drug used in this study, gastrointestinal side effects were commonly reported. Basic science studies: Pentoxifylline (POF), a non-specific phosphodiesterase inhibitor, may have beneficial effects in patients with sarcoidosis, via its known inhibition of the production of cytokines such as tumor necrosis factor-alpha (TNF-alpha) and IL-12. Human blood monocyte-derived macrophages from normal volunteers were incubated with and without E. coli 0128:B12 lipopolysaccharide (LPS) (1ug/mL) and 1mM POF for 18 hr, followed by isolation of RNA for hybridization with oligonucleotide-based microarrays. Among mRNAs that were increased at least 2-fold by LPS and significantly inhibited by POF (P < 0.01) were: interferon-stimulated protein, 15kDa (ISG15), epithelial membrane protein 3 (EMP3), 2?-5?-oligoadenylate synthetase 3, 100kDa (OAS3), bone marrow stromal cell antigen 2 (BST2), and IPA proteasome (prosome, macropain) subunit, beta type 9 (PSMB9). In particular, LPS increased expression of ISG15 8.2-fold, which was reduced only to 4.4-fold control values by POF (P< 0.005). Real-time quantitative PCR confirmed the partial reversal by POF of the stimulatory effect of LPS on expression of ISG15. ISG15 may be a relevant target in sarcoidosis since it is known that induction of ISG15 by IFN-alpha results in increased IFN-alpha, a key Th1 cytokine associated with granulomatous responses. Interestingly, LPS failed to induce ISG15 in alveolar macrophages from normal volunteers, which suggests that ISG15 expression may be differently regulated in alveolar macrophages and macrophages derived from blood monocytes. In another study, we found that cAMP-elevating agents, prostaglandin E1 and cholera toxin (which activate adenylyl cyclase via distinct mechanisms), and the cyclic nucleotide phosphodiesterase (PDE) inhibitors, cilostamide (PDE3 inhibitor) and rolipram (PDE4 inhibitor), induced apoptosis in FDCP2 promyeloid cells. The cAMP analogs, 8-bromo-cyclic AMP and Sp-cAMP, also induced apoptosis in these cells. The PKA inhibitor, 8-bromo-Rp-cAMPS, or insulin-like growth factor-1 (which activates both PDE3 and PDE4 in FDCP2 cells), prevented apoptosis induced by cAMP-elevating agents. The pro-apoptotic protein, Bax, was redistributed from cytosol to membranes in apoptotic cells. This redistribution could be prevented by treatment of cells with IGF-1. Removal of serum from culture medium led to dephosphorylation of Erk1/2 and PKB in FDCP2 cells, and cAMP-elevating agents significantly accelerated this process. Activation of phosphatases was found to bbe more important in dephosphorylation of Erk1/2 than inhibition of upstream protein kinases. Our results suggest that in FDCP2 cells, cAMP-elevating agents, acting through PKA and PP2A or PP2A-like protein phosphatases, inhibit Raf-MEK-Erk and PKB signaling and thereby abrogate Erk-and PKB-mediated survival signals, with concurrent translocation of Bax to membranes, perhaps resulting in Bax-mediated apoptosis. On the other hand, in these cells, IGF-1 promotes anti-apoptotic or survival signals, most likely by two mechanisms. IGF-1 activates ERK and PKB via phosphatidyl-inositol-3 kinase (PI3-K) dependent pathways, thus promoting anti-apoptotic signals, which include activation of both PDE3 and PDE4, leading to a reduction in cAMP and consequent inhibition of anti-proliferative and pro-apoptotic effects of cAMP. Taken together, these studies support the hypothesis that, in FDCP2 cells, PDEs function as anti-apoptotic, and protein phosphatases as pro-apoptotic, regulators.