Phosphodiesterases as Therapeutic Targets: Sarcoidosis

临床研究：抑制与肉芽肿形成和维持相关的炎性细胞因子的类固醇节约药物将是结节病管理中非常有用的治疗方法。在这方面，已报道喷替福林（POF）抑制IL-2、IL-12、TNF-α的产生，并且在开放标签临床研究中，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治疗是否会改善或稳定肺结节病，以及作为皮质类固醇治疗的辅助治疗是否有益。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。虽然肺结节病复发不被认为是正式的终点，但POF治疗组5/13例患者发生疾病复发，而安慰剂治疗组12/14例患者发生疾病复发（P < 0.02）。安慰剂组无自发缓解。24周后，POF治疗组的平均泼尼松剂量显著低于安慰剂对照组（P < 0.006）。不良反应，主要是胃肠道，报告了11/13 POF治疗的患者，但没有安慰剂治疗的患者。因此，虽然POF可能对肺结节病有皮质类固醇节省作用，但在本研究中使用的药物浓度下，胃肠道副作用通常被报道。 基础科学研究：非特异性磷酸二酯酶抑制剂喷替福林（POF）可能通过其已知的抑制细胞因子（如肿瘤坏死因子-α（TNF-α）和IL-12）的产生，对结节病患者产生有益作用。将正常志愿者的人血单核细胞衍生的巨噬细胞与E. coli 0128：B12脂多糖（LPS）（1 μ g/mL）和1 mM POF处理18小时，然后分离RNA用于与基于阿托伐他汀的微阵列杂交。在LPS刺激后增加2倍以上而POF明显抑制（P < 0.01）的mRNA中，有干扰素刺激蛋白15 kDa（ISG 15）、上皮膜蛋白3（EMP 3）、2？五个？寡腺苷酸合成酶3，100 kDa（OAS 3），骨髓基质细胞抗原2（BST 2），和IPA蛋白酶体（前体，巨蛋白酶）亚基，β 9型（PSMB 9）。特别地，LPS使ISG 15的表达增加8.2倍，其通过POF仅降低至对照值的4.4倍（P< 0.005）。实时定量PCR证实了部分逆转POF的LPS对ISG 15表达的刺激作用。ISG 15可能是结节病中的相关靶标，因为已知IFN-α诱导ISG 15导致IFN-α增加，IFN-α是与肉芽肿反应相关的关键Th 1细胞因子。有趣的是，LPS未能在正常志愿者的肺泡巨噬细胞中诱导ISG 15，这表明ISG 15表达在肺泡巨噬细胞和来源于血液单核细胞的巨噬细胞中可能受到不同的调节。 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（通过不同的机制激活腺苷酸环化酶）以及环核苷酸磷酸二酯酶（PDE）抑制剂西洛酰胺（PDE 3抑制剂）和咯利普兰（PDE 4抑制剂）诱导FDCP 2早幼粒细胞凋亡。cAMP类似物，8-溴环AMP和Sp-cAMP，也诱导这些细胞的凋亡。PKA抑制剂8-溴-Rp-cAMPS或胰岛素样生长因子-1（可激活FDCP 2细胞中的PDE 3和PDE 4）可阻止cAMP升高剂诱导的细胞凋亡。促凋亡蛋白Bax在凋亡细胞中从胞浆重新分布到细胞膜。这种再分配可以通过用IGF-1处理细胞来防止。从培养基中去除血清导致FDCP 2细胞中Erk 1/2和PKB的去磷酸化，cAMP升高剂显著加速了这一过程。磷酸酶的激活被认为比上游蛋白激酶的抑制在Erk 1/2的去磷酸化中更重要。我们的研究结果表明，在FDCP 2细胞中，cAMP升高剂，通过PKA和PP 2A或PP 2A样蛋白磷酸酶，抑制Raf-MEK-Erk和PKB信号，从而废除Erk和PKB介导的生存信号，与Bax的同时易位到膜，可能导致在ERK介导的细胞凋亡。另一方面，在这些细胞中，IGF-1促进抗凋亡或存活信号，最有可能通过两种机制。IGF-1通过磷脂酰-肌醇-3激酶（PI 3-K）依赖性途径激活ERK和PKB，从而促进抗凋亡信号，其包括PDE 3和PDE 4的激活，导致cAMP减少并随后抑制cAMP的抗增殖和促凋亡作用。总之，这些研究支持了以下假设：在FDCP 2细胞中，PDE起抗凋亡调节剂的作用，蛋白磷酸酶起促凋亡调节剂的作用。

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.